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Retroviruses 2004: Review of the 2004 Cold Spring Harbor Retroviruses conference

For the past several decades, retrovirologists from around the world have gathered in late May at the Cold Spring Harbor Laboratories in New York to present their studies in formal talks and posters, and to discuss their ongoing research informally at the bar or on the beach. As organizers of the 2004 Cold Spring Harbor Retroviruses Conference, we have been asked by the editors of Retrovirology to prepare a review of the meeting for publication on-line. Our goal in this review is not to provide a detailed description of data presented at the meeting but rather to highlight some of the significant developments reported this year. The review is structured in a manner that parallels the organization of the meeting; beginning with the entry phase of the replication cycle, proceeding with post-entry events, assembly and release, integration, reverse transcription, pathogenesis/host factors, RNA-related events (transcription, processing, export, and packaging) and finishing with antivirals. While the most striking developments this year involved post-entry events and assembly/release, significant progress was made towards elucidating a number of aspects of the retroviral replication cycle.

Entry
Although no "new" retrovirus receptors were reported at the meeting, several talks centered on recently discovered receptors. N. Manel, from the groups that identified GLUT-1 as an entry receptor for HTLV-1 (N. Taylor, J.-L. Battini, and M. Sitbon) [1], proposed that part of the pathogenic effects of this virus may be due to its perturbation of glucose metabolism. HTLV-1-infected tissue culture cells display decreased glucose uptake as a result of envelope (Env) glycoprotein-GLUT-1 interaction. The authors speculated that if this disruption of glucose metabolism also occurs in vivo, it might provide insights into the neuronal damage that occurs in some HTLV-1-infected patients. They also suggested that Env-mediated impair-ment of GLUT-1 function might contribute to the emergence of preleukemic T cells with new selective advantages [2].
The co-receptor for feline immunodeficiency virus (FIV), perhaps the best non-primate model for HIV-1, has also recently been identified. Work presented by J. Elder showed that FIV preferentially infects certain subsets of T cells through interaction with CXCR4 and a 43 kDa protein. This 43 kDa protein turns out to be CD134, recently demonstrated to be a receptor for FIV [3]. CD134 was first described as a member of the tumor necrosis/nerve growth factor receptor family expressed on activated T cells. By analogy with the role of CD4 in HIV infection, CD134 may target FIV infection to a particular subset of T cells. Extending the CD4/HIV parallel, CD134 may be the molecule that initially engages FIV, followed by CXCR4 binding and virus/cell fusion. Elder suggested that CD134 should be referred to as the FIV attachment receptor and CXCR4 as the entry receptor.
The use of alternative chemokine co-receptors by primary HIV-1 isolates was discussed by S. Neil from R. Weiss's group. They proposed that some primary dual-tropic HIV-1 isolates, especially those isolated from early seroconverters, might infect primary astrocytes, endothelial cells and macrophages through the use of D6, a promiscuous chemokine receptor highly expressed on these cell types. They hypothesized that D6 usage to infect endothelial cells could influence colonization of endothelial compartments and promote placental transmission.
Two groups discussed the risk of pig endogenous retrovirus (PERV) infection of human cells as a potential problem for xenotransplantation. I. Harrison from the Stoye lab showed that recombination between different endogenous PERVs (A and C), which normally have very low titers on human cells, could lead to the production of virus with the capacity to efficiently infect human cells. This tropism change mapped in large part to the Env glycoprotein, but the Gag-Pol region was also implicated. D. Lavillette from the Kabat lab presented evidence that wild-type PERVs, or mutants lacking fully infectious envelopes due to alterations in a conserved PHQ motif (in SU) required for γ-retrovirus infection, could bind and enter human cells when added together with the envelope from gibbon-ape leukemia virus, which does infect human cells. The ability of functional Env glycoproteins from infectious viruses to trans-complement infection by viruses with mutant envelopes or with restricted tropism on particular target cells has been reported for several retroviruses [4,5]. In the case of PERVs, such complementation has the potential of overcoming host-range and interference barriers and could pose a hazard for xenotransplantation.
For a number of years, investigators have attempted to engineer peptide ligands for cell surface receptors into retroviral Env glycoproteins with the goal of designing retroviral gene therapy vectors that would efficiently target specific cell types. These attempts have been hampered by low transduction efficiencies. L. Albritton presented work from her lab demonstrating that incorporation of a peptide sequence into the SU receptor binding site (RBS) may overcome this problem. Her lab constructed a chimera in which the RBS of the Moloney murine leukemia virus (MLV) Env was replaced with the peptide ligand somatostatin (Sst). Such chimeras efficiently infected human cells expressing the Sst receptor, but could no longer infect mouse cells expressing the natural receptor, ATCR1. The similarity in length of the peptide with the sequence it replaced, as well as the structural similarity between ATCR1 and the Sst receptor, may have contributed to the success of this approach, since substitution of the RBS with human stromal-derived factor-1 alpha (SDF-1α), which also uses a structurally similar receptor (CXCR4), has also been successful [6]. In contrast, attempts to make peptide ligand substitutions in this RBS that are either dissimilar in length or use structurally unrelated receptors have met with limited success (i.e. see [7].
Following binding between retroviral Env glycoproteins and their receptors, conformational changes must take place in both the SU and TM that expose the fusion peptide and enable membrane fusion to occur. H. Garoff presented work extending his recently published report [8] showing that for γ-retroviruses this conformational change involves SU-TM disulfide bond isomerization. The isomerization, which is inhibited by Ca 2+ , results in breakage of the SU-TM disulfide bond, the generation of an intra-SU bond, and subsequent exposure of the TM fusion peptide. Garoff speculated that the ability of γ-retroviruses such as MLV to fuse at the cell surface, in contrast to α-retroviruses like ALV that lack isomerization activity and which apparently undergo pH-dependent fusion in a subcellular compartment, is the result of this isomerization.
Several studies have implicated amino acid changes in the cytoplasmic domain of the MLV TM on SU conformation, with consequent effects on viral infectivity and antibody recognition [9,10]. Work presented by R. Montelaro demonstrated that mutations in the cytoplasmic tail of the HIV-1 TM (gp41) can also result in escape from neutralization. Importantly, at least one of these antibody-escape mutants still retained wild-type infectivity, indicating that sequence changes in the TM intracytoplasmic domain should be considered in the characterization of antigenic variants of HIV-1 that escape neutralization.
Dendritic cells express a molecule known as DC-SIGN that binds HIV-1 particles and facilitates their transmission to susceptible target cells [11]. The mechanism by which this molecule promotes the transfer of infectious virions to target cells has been an active area of investigation for the past several years. Work from V. KewalRamani's lab (Wu et al.) showed that DC-SIGN is able to transfer HIV-1 infectivity in trans when expressed on some cell types but not when expressed on others. The ability of DC-SIGN to promote virus transfer appears to correlate with the localization of the particles on the DC-SIGNexpressing cell; in cells unable to mediate transfer, virions are internalized, whereas cells able to mediate efficient transfer retain virions at the cell surface. Wu and col-leagues also observed that binding and transmission of HIV-1 by immature dendritic cells is Env-and DC-SIGNdependent, whereas virus binding by mature dendritic cells does not require either Env on the particle or DC-SIGN on the dendritic cell. The molecule(s) expressed on dendritic cells that potentiate DC-SIGN-independent virus transmission await discovery.

Post-entry events
The last two years have seen major advances in our understanding of the mechanisms by which cells from human and non-human primates restrict retroviral infection. In 2002, Sheehy et al. [12] reported that the HIV-1 accessory protein Vif interacts with the cellular cytidine deaminase APOBEC3G. In the absence of Vif expression, APOBEC3G is incorporated into HIV-1 virions and, during reverse transcription in the target cell, converts cytosines to uracils. This conversion results in the degradation of newly synthesized DNA through the action of host glycosidases and repair enzymes, and leads to G-to-A hypermutation in the newly synthesized viral DNA. Vif counters the antiviral activity of APOBEC3G by blocking its incorporation into virions in the producer cell. Interestingly, Vif displays species specificity in its ability to inactivate APOBEC3G; for example, HIV-1 Vif blocks the antiviral activity of human but not African green monkey APOBEC3G, and SIVagm Vif inactivates African green monkey but not human APOBEC3G. The labs of N. Landau and V. Pathak both reported that the determinant of this species specificity maps to amino acid 128 of APOBEC3G. Data in the Landau lab presentation (Schrofelbauer et al.) suggested that the inability of HIV-1 Vif to block the activity of African green monkey APOBEC3G was due to a lack of binding between these two proteins, whereas the Pathak lab (Xu et al.) reported that mutation of residue 128 of human APOBEC3G did not prevent binding of the mutant protein to Vif. Both labs recently published their findings [13,14].
Since the incorporation of APOBEC3G into virus particles is required for its antiviral activity, several groups have focused on how this cellular protein is incorporated. The mechanism of APOBEC3G incorporation is of particular interest since it appears not to be specific for HIV-1 (e.g., human APOBEC3G is incorporated into MLV particles) and because inhibitors that disrupt Vif's ability to block APOBEC3G incorporation would presumably display antiviral properties. L. Kleiman's lab (Cen et al.) reported that APOBEC3G interacts directly with Gag in a manner dependent on the Gag nucleocapsid (NC) domain. The labs of W. Popik and X.F. Yu also observed that NC plays an important role in the APOBEC3G/Gag interaction. H. Xu from V. Pathak's group reported that mutation of NC reduced but did not abrogate APOBEC3G incorporation into virions. Since NC is a major determinant of RNA packaging into virions, these data, together with the above-mentioned finding that APOBEC3G is incorporated into MLV particles, can be rationalized by a model that proposes an important role for RNA (either viral or cellular) in APOBEC3G incorporation. Such a model would also be consistent with the apparent inability of HIV-1 to evade APOBEC3G incorporation simply by mutation of a specific protein-protein binding site. Interestingly, a study from D. Ho's lab (Simon et al.) found that isolates of HIV-1 that are unable to block APOBEC3G activity are relatively common in infected patients. The authors suggested that sporadic Vif inactivation might be a factor in promoting viral evolution in vivo since Vifdefective variants would exhibit a higher mutation rate.
In recent years, several groups have reported that HIV-1 is unable to efficiently infect cells from certain species of Old World monkey (for review see [15]). The block is imposed early post-entry, prior to reverse transcription, and is mediated by an inhibitory factor expressed in Old World monkey cells. The viral determinant of this restriction maps to the capsid (CA) domain of Gag, making this restriction somewhat reminiscent of the Fv1 block described by Lilly and others decades ago [16]. J. Sodroski's group recently published that the rhesus macaque version of the cytoplasmic body component TRIM5α [17] potently blocks HIV-1 infection. Presentations from the labs of J. Sodroski (Stremlau et al.) and P. Bieniasz (Hatziioannou et al.) reported that Ref1 and TRIM5α are species-specific variants of TRIM5α; this finding has now been published [18]. This factor is quite distinct from Fv1, which bears sequence homology to MLV CA [19]. A. Lassaux (from the Battini and Sitbon labs) reported that Fv1 and Ref1 recognize different amino acid combinations within the same 100 amino acid determinant of the MLV CA. Hatziioannou et al. also described their results on characterizing the ability of human-and monkey-derived TRIM5α's to block the entry of a panel of retroviruses. D. Sayah from J. Luban's lab provided a preview of the now-published paper [20] reporting the intriguing finding that in owl monkey cells, the post-entry block to HIV-1 infection is conferred by a TRIM5α-cyclophilin A fusion protein. In another presentation focused on post-entry blocks to retroviral infection, V. KewalRamani's lab (Martin et al.) reported that overexpression of a truncated form of an RNA binding protein that is a component of the poly A machinery blocks an early stage of the HIV-1 infection process without disrupting MLV infectivity.
J. Young (Narayan and Young) reported the development of a cell-free uncoating assay that will likely prove useful in defining the role of cellular factors in stimulating or blocking early post-entry steps in the replication cycle. Avian sarcoma/leukosis virus (ASLV) particles can be trapped in endosomes when cells expressing a GPI-linked version of the ASLV receptor are infected in the presence of NH 4 Cl. The virus-containing endosomes can then be isolated; upon removal of the NH 4 Cl in vitro, fusion and reverse transcription take place in a manner dependent upon ATP hydrolysis and the presence of cellular factors. This cell-free uncoating assay can be adapted to other viruses (e.g., HIV-1) by pseudotyping with the ASLV Env glycoprotein. The authors reported that monkey cell restriction factors, and cyclosporin A (which blocks cyclophilin A incorporation into HIV-1 particles), inhibit reverse transcription in this system, suggesting that it faithfully recapitulates certain key aspects of uncoating in infected cells. Some of the data in this presentation were recently published [21].

Assembly and Release
Major developments have taken place in the past several years in our thinking about the location in the host cell at which retrovirus assembly takes place, and the mechanism by which Gag proteins target the subcellular site of assembly. Until recently, it was felt that viruses that follow the type C assembly pathway [including the lentiviruses (e.g., HIV-1), δ-retroviruses (e.g., HTLV-1), γ-retroviruses (e.g. MLV), etc.] assemble at the plasma membrane. However, it has long been recognized that in some cases (for example, HIV-1 in macrophages) assembly and budding take place at an intracellular compartment. Several groups have recently demonstrated that this compartment is the late endosome or multivesicular body (MVB), and it now appears that while the plasma membrane likely represents the predominant site of assembly for these viruses, Gag can also target and assemble in an endosomal compartment in a variety of cell types. M. Resh's lab (Perlman and Resh) used the tetracyteine/biarsenical live-cell labeling system reported by Gaietta et al. [22] to follow Gag trafficking relatively early (1-4 hrs.) post-synthesis. Their results suggest that Gag may first localize to secretory lysosomes and then subsequently "ride" these vesicles to the plasma membrane. M. Thali's lab recently reported that a significant fraction of HIV-1 Gag colocalizes with late endosomal markers both at intracellular membranes and at the plasma membrane [23]. Together with D. Ott's lab (Nydegger et al.), they have also started using the tetracysteine/biarsenical technology to analyze Gag localization. Like the Resh lab, they observe some association of Gag with late endosomal/MVB membrane, but a significant fraction of newly synthesized Gag appears to associate directly with discrete plasma membrane microdomains. P. Spearman's lab (Dong et al.) reported interesting findings implicating the AP-3 clathrin adaptor protein complex (which among other things regulates CD63 trafficking) in HIV-1 Gag targeting. They observed that the δ subunit of AP-3 interacts with the MA domain of Gag and that either overexpression of an N-terminal δ subunit fragment or siRNA knockdown of AP-3 δ subunit expression inhibits HIV-1 release.
H. Wang from L. Mansky's group observed that HTLV-1 assembly, like that of HIV-1, can take place in the MVB. Interestingly, while in HeLa cells HTLV-1 assembly appears to occur primarily at the plasma membrane, mutational disruption of the Pro-Thr-Ala-Pro (PTAP) late domain results in accumulation of virus particles in the MVB, consistent with published results (e.g., [24]). T. Hope's lab (Gomez and Hope) confirmed the finding [25] that HIV-1 mutants lacking the p6 late domain still assemble in MVBs in macrophages, indicating that interactions between p6 and cellular host factors (e.g., Tsg101) are not required for MVB localization. Reports from the labs of D. Muriaux (Grigorov et al.) and F.-L. Cosset (Sandrin et al.) suggested that interactions between MLV Gag and Env may occur in an intracellular compartment and that this interaction might influence both Gag trafficking and Env incorporation into virions. Some of this work has now been published [26].
A study from the Freed lab (Ono and Freed) examined the possibility that the phosphoinositide phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ) plays a role in Gag targeting. This lipid is of interest since it is involved in the trafficking of a variety of cellular proteins that, like many retroviral Gag proteins, contain a basic membrane binding domain. Furthermore, work from A. Rein's lab [27] has shown that, in vitro, HIV-1 Gag binds molecules structurally related to phosphoinositides. A. Ono used enzymatic approaches to perturb cellular PI(4,5)P 2 levels in virus-expressing cells and observed that Gag targeting and virus assembly were shifted from the plasma membrane to intracellular compartments. These results indicate that cellular PI(4,5)P 2 plays a role in directing Gag to the plasma membrane.
Work from J. Lingappa's lab previously suggested the involvement of the cellular RNase L inhibitor HP68 in HIV-1 assembly [28]. This work was extended at the meeting by Dooher et al., who showed that HP68, Gag, and genomic RNA colocalize in virus-producing cells. The cellular protein nucleolin was also found to be a component of the Gag/HP68 complex.
Novel findings relating to the regulation of foamy virus (FV) release were reported by the Lindemann lab. FVs are unusual among retroviruses in that Env glycoprotein expression plays a critical role in particle release. The leader peptide of FV Env, which is generated by a cleavage event during Env trafficking to the plasma membrane and is incorporated into particles, seems to play an important role in the budding process. Lindemann et al. reported the identification of ubiquitylated forms of the leader pep-tide; suppression of these ubiquitylated forms markedly stimulated subviral particle release. These results suggest that ubiquitylation of the FV Env leader peptide modulates the ratio of particle vs. subviral particle budding.
Progress continues to be made in visualizing retrovirus assembly and release in real time in living cells. In addition to use of the tetracyteine/biarsenical live-cell labeling system described above, B. Muller from H.-G. Krausslich's lab discussed the development of an infectious HIV-1 derivative containing a GFP insert near the C-terminus of MA. This derivative produces particles with wild-type morphology; however, release kinetics are impaired. While the Gag-GFP virus is poorly infectious, infectivity can be restored upon coexpression with wild-type HIV-1. This Gag-GFP HIV-1 derivative should be useful for both assembly/release and post-entry studies. In a presentation from the labs of V. Vogt and W. Webb, D. Larson described the use of correlated fluorescence microscopy and scanning electron microscopy to visualize Rous sarcoma virus (RSV) budding in real time using a GagGFP derivative.
Major advances have been made in the past three years in elucidating the host cell machinery required for the release of retrovirus particles from infected cells [29,30]. It is now well accepted that retroviruses use their late domains to commandeer machinery that normally plays a central role in promoting the budding of vesicles into the MVB. This machinery includes the so-called "class E Vps" factors originally identified in yeast as being crucial for MVB biogenesis [31]. Many of these class E Vps proteins are found in three multisubunit complexes known as ESCRT-I, -II, and -III. Retroviral late domains come in three flavors: Pro-Thr/Ser-Ala-Pro [P(T/S)AP], Pro-Pro-x-Tyr (PPxY), and Tyr-Pro-Asp-Leu (YPDL). HIV-1 release is controlled predominantly by a P(T/S)AP-type late domain; many retroviruses, including MLV and RSV, contain PPxY late domains; and equine infectious anemia virus (EIAV) harbors a YPDL late domain. Several retroviruses [e.g., Mason-Pfizer monkey virus (M-PMV) and HTLV-1] encode both P(T/S)AP and PPxY late domains. In addition to its P(T/S)AP motif, HIV-1 contains a secondary YPDL-related sequence whose role in HIV-1 release remains to be defined. It is now well established that the P(T/S)AP motif interacts with the ESCRT-I component Tsg101. Recent data from several labs (those of H. Gottlinger, W. Sundquist, and P. Bieniasz) have strongly suggested that AIP1 (also known as ALIX) is the host factor with which YPDL interacts. There is less certainty about the identity of the biologically relevant PPxY-interacting protein. PPxY motifs in cellular proteins often interact with WW domains, and several PPxY-containing retroviral Gag proteins have been reported to bind the ubiquitin ligase Nedd4 (or related proteins), which con-tains a series of centrally located WW domains. P. Bieniasz's lab (Martin-Serrano et al.) reported at the meeting that MLV Gag binds the Nedd4-related proteins WWP1 (which has also been shown to associate with HTLV-1 Gag [32]), WWP2 and ITCHY. The extent to which binding to these proteins was reduced by mutations in the PPPY motif correlated with the severity of the budding defect induced by the mutations. The authors also observed that WWP1 localizes to aberrant endosomes induced by expression of a dominant-negative Vps4 in a manner dependent on the ubiquitin ligase (or HECT) domain, suggesting that the HECT domain may link WWP1 (and consequently Gag) to the class E Vps machinery. J. Leis's lab previously reported that overexpression of the WW domain-containing region from a chicken Nedd4-like protein inhibited RSV particle production. At In yeast, ESCRT-I contains three protein components: Vps23 (the yeast homolog of Tsg101), Vps28 and Vps37. In mammalian cells, only the Vps23 and Vps28 homologs had been identified. At this year's meeting, M. Stuchell from W. Sundquist's lab reported the cloning of human Vps37. As in yeast, human Vps37 binds Tsg101 and is present along with Tsg101 and Vps28 in a high molecular weight ESCRT-I complex. Much of this work has recently been published [33], as have similar results from H. Stenmark's lab [34]. Further illustrating the importance of ESCRT-I in HIV-1 budding, fusion of Vps37 to the C-terminus of Gag reverses the release defect imposed by PTAP deletion [33]. Y. Yardin's lab (Amit et al.) described the identification of an E3 ubiquitin ligase (termed Tal, for Tsg101-associated ligase) that binds the N-terminal UEV domain of Tsg101 and apparently regulates its activity. This work has also recently been published [35].
M. Palmarini's lab (Mura et al.) described a novel type of retroviral interference that operates at the level of virus assembly and release. The sheep genome harbors a number of copies of endogenous retroviruses closely related to the pathogenic exogenous β-retrovirus Jaagsiekte sheep retrovirus (JSRV). One of these endogenous retroviruses (enJS56A1) displays a defect in assembly/release. EM analysis indicates that enJS56A1 particles form large perinuclear aggregates; these appear to trap JSRV particles intracellularly when enJS56A1 and JSRV are coexpressed. The authors speculate that enJS56A1 may have helped protect sheep from infection with related, exogenous retroviruses during evolution. Much of this study has recently been published [36].
It has been known for many years that several retroviruses express two forms of Gag: a conventional version and a larger, glycosylated form referred to as glycoGag. Gly-coGag is synthesized using an alternative, upstream initiation codon, and unlike its smaller counterpart it is transported to the plasma membrane through the secretory pathway. H. Fan's lab (Low et al.) reported at this year's meeting that a packaging cell line that expresses Gag without glycoGag produces tube-shaped particles at the cell surface. This apparent assembly defect was corrected by coexpression of glycoGag. The expression of glycoGag increased both virus yield and infectivity. These results suggest that glycoGag may function, in a manner that remains to be elucidated, in regulating proper virus assembly and release.

Integration
Retroviral preintegration complexes (PICs) and the integrase (IN) enzyme itself, interact with a variety of host factors during nuclear import of the PIC and integration of the newly synthesized viral DNA into the host cell chromosome. One such host factor, lens epithelium-derived growth factor (LEDGF/p75), was recently reported to bind HIV-1 IN [37,38]. Several presentations at this year's meeting provided varied and conflicting results regarding the role of LEDGF/p75 in the integration process. S. Emiliani reported data from a collaboration between the labs of R. Benarous and Z. Debyser showing that knockdown of LEDGF/p75 expression using siRNA in HeLa P4 cells inhibited HIV-1 replication without affecting the import of IN to the nucleus. The Q168L mutation in HIV-1 IN abolished both virus replication and the interaction between IN and LEDGF/p75. Busschots from the Debyser lab showed that the interaction of LEDGF/p75 with IN increased the affinity of IN for DNA. Based on these data, it was postulated that LEDGF/p75 may play a role in tethering IN to chromosomal DNA. E. Poeschla's lab (Llano et al., also see [39]) observed that endogenous LEDGF/p75 co-immunoprecipitated with both HIV-1 and FIV IN. Stable knock-down of LEDGF/p75 expression in 293T cells induced a redistribution of both HIV-1 and FIV IN from the nucleus to the cytoplasm but apparently did not affect nuclear import of HIV-1 or FIV PICs since lentiviral vector infectivity was not reduced under these conditions. Furthermore, stable knock-down of LEDGF/p75 expression in the Jurkat T-cell line did not affect HIV-1 replication. However, LEDGF/p75 was found to be a component of lentiviral PICs. The authors concluded that LEDGF/p75 is not required for lentiviral integration but advanced the hypothesis that it might play a role in target site selection. Vandekerckhove and colleagues from the Debyser lab reported that stable knock-down of LEDGF/p75 expres-sion in HeLa P4 or MOLT cells delayed HIV-1 replication but did not diminish infectivity mediated by a VSV-G pseudotyped lentiviral vector. The authors controlled for non-specific siRNA effects by using double mismatched siRNA. Results from A. Engleman and coworkers (Vandegraaff et al.) raised further questions concerning the role of LEDGF/p75 in HIV-1 integration. They observed that while two LEDGF/p75 siRNAs reduced LEDGF/p75 protein levels, only the one originally described by the Debyser lab impaired HIV-1 infectivity. This infectivity defect could not be reversed by partially restoring LEDGF/ p75 expression. Based on these results, the authors cautioned that the effect of LEDGF/p75 siRNA on HIV-1 infectivity may be due to non-specific effects not directly related to LEDGF/p75. Clearly, additional studies need to be performed to clarify the role of LEDGF/p75 in lentiviral integration.
A number of presentations focused on the target site specificity of retroviral (or retrotransposon) integration. Different retroviruses and retroelements display strong preferences in selecting their target sites in the genomes of their host cells (for review see [40]). For example, the Ty1 and Ty3 retrotransposons integrate predominantly upstream of Pol III-transcribed genes; the Tf1 retrotransposon selects sequences upstream of Pol II-transcribed genes; HIV-1 tends to integrate in actively transcribed regions; and MLV prefers to integrate in the promoters of active genes. At this year's meeting, A. Narezkina from R. Katz's and A. Skalka's lab, and R. Mitchell from F. Bushman's group, both described the results of genome-wide analyses of ASLV integration sites. They observed that, unlike MLV, this avian retrovirus does not prefer to integrate at transcription start sites, and unlike HIV-1 does not display a strong preference for highly active genes. Both groups did observe a relatively modest tendency for integration within genes. H. Levin's group (Kelly et al.) extended their previous work on Tf1 integration. Using a target plasmid containing a single gene, they observed that nearly all integration events took place in the gene's promoter region. Interestingly, the integration sites displayed a periodic pattern in which integrated copies of Tf1 were separated by around 30 nucleotides. This targeting appeared to be dependent on promoter activity. Kelly and coworkers speculated that the chromodomain in the Tf1 IN binds histones and regulates integration into specific targets in a promoter-positioned nucleosome. Holman and Coffin reported on the analysis of base preferences immediately surrounding integrated HIV-1 proviruses. Using a large amount of data derived from previously reported integration sites, the authors observed strong base preferences within seven residues at either end of integrated proviruses. The presentation emphasized that HIV-1 integration shows target preferences on both a macro scale (as noted above) and on a microscale, involv-ing the residues immediately adjacent to the integration site.

M. Katzman's lab (Konsavage et al.) reported an interest-
ing RSV IN mutant that displayed enhanced 3'-end processing activity but impaired DNA joining. The authors speculated that RSV IN has evolved suboptimal processing activity to allow it to catalyze DNA joining.
R. Craigie's lab previously reported that a protein termed "barrier-to-autointegration factor" (BAF) is a component of the MLV preintegration complex and that this factor enhances intermolecular integration reactions and blocks intramolecular integration (autointegration) [41]. At this year's meeting, Suzuki and Craigie extended this work by showing that BAF interacts with an inner nuclear membrane protein, lamina-associated polypeptide 2α (LAP2α). LAP2α is a component of the MLV preintegration complex and knockdown of its expression inhibits MLV replication.

Reverse Transcription
A number of studies over the years have demonstrated that retroviral NC proteins possess nucleic acid chaperone activity that plays an important role in various aspects of reverse transcription (for review, see [42]). Several presentations focused on this activity of NC. Work from J. Levin's lab (Guo et al.) demonstrated that in an in vitro assay that models minus-strand transfer, NC alone is able to catalyze the removal of small 5' terminal genomic RNA fragments, which remain annealed to a minus-strand strong-stop DNA. Strand transfer product increased with increasing NC, and in the presence of NC, strand transfer product was generated even when reverse transcription was catalyzed by an RNase H-deficient RT. The NC zinc fingers appeared to be critical for this activity. These results suggest an important role for NC nucleic acid chaperone activity in removing terminal RNA fragments annealed to minus-strand strong-stop DNA following primary cleavage by RNase H. By examining reverse transcription in infected cells, R. Gorelick's lab (Thomas et al.) observed that mutations in the first zinc finger of NC strongly interfered with the progression of reverse transcription and impaired virus infectivity. The authors speculated that reduced binding of NC to the viral DNA allows cellular enzymes (nucleases and ligases) to modify the viral DNA ends thereby interfering with integration.
Reverse transcription is initiated by a host cell-derived tRNA bound to the primer binding site (PBS) near the 5' end of the viral genome. Retroviruses are selective in their utilization of host tRNAs; HIV-1, for example, specifically primes reverse transcription with a tRNA lys3 . The selectivity for particular tRNAs results from interactions between the tRNA and the PBS and it has been proposed by B.
Berkhout and colleagues that a second motif in the viral RNA, termed the primer-activation signal (PAS), also forms specific contacts with the tRNA. Berkhout's lab (Abbink et al.) reported at this year's meeting that, by mutagenesis of the PBS and PAS, HIV-1 primer utilization could be shifted from tRNA lys3 to tRNA lys1,2 . Such mutants replicated poorly but could adapt during long-term passaging. In one case, adaptation evidently occurred through optimization of the putative PAS. Interestingly, a single amino acid change in the RNase H domain of RT also arose during adaptation, suggesting a possible role for RT in selective primer utilization.
While PCR techniques allow the progression of viral DNA synthesis to be monitored post-infection, it has not been possible to follow reverse transcription in a strand-specific fashion in infected cells. D. Thomas from V. Pathak's lab reported the development of a strand-specific amplification assay that uses so-called "padlock" probes -long single-stranded oligonucleotides that hybridize with their target sequence simultaneously at their 5' and 3' ends. Following hybridization, the ends of the padlock probes are ligated to form circles that are amplified and detected by real-time PCR. This assay, which allows specific steps in reverse transcription to be measured quantitatively in a strand-specific manner, should be very useful for addressing a number of questions regarding the kinetics of reverse transcription and the efficiency of this process under a variety of conditions. F. Maldarelli described the results of studies conducted with S. Palmer, J. Coffin and colleagues aimed at characterizing the evolution of HIV-1 populations in vivo. The authors monitored genetic variation in cohorts of drugnaïve and drug-resistant patients by analyzing individual pro-pol sequences. In both drug-naïve and drug-resistant patients, they observed little change in virus population structure over several years, implying that the replicating population is relatively large. Interestingly, recombination rates appeared to be very high regardless of levels of viremia, suggesting the presence of a substantial number of multiply infected cells even at low viral loads. The results emphasize the importance of recombination in generating viral diversity in vivo.

Pathogenesis
The keynote speaker at this year's meeting was Neal Copeland, who presented important findings from his and Nancy Jenkins' lab regarding the use of high through-put analysis to map retroviral integration sites in tumors induced by MLVs. This rapidly developing approach is being used by a number of groups to elucidate cancer gene pathways [43][44][45][46], and to define retroviral integration site preferences. The Jenkins and Copeland labs have used infection of various hematopoietic stem cells followed by transplantation into lethally irradiated mice to identify not only novel proto-oncogenes, but also cooperating cancer genes, tumor suppressors, and genes involved in stem cell transformation and immortalization. While this technique is currently applicable only to hematopoietic lineage cells, current research in the Jenkins and Copeland labs is testing whether integration by transposable elements in other cell types can be used to identify similar genes and pathways in solid tumors.
In contrast to the majority of leukemia-inducing retroviruses in non-human species, most of which cause cancer by insertional mutagenesis, HTLV-I encodes accessory proteins, such as Tax, with known transforming activity. There were several talks at the meeting describing new accessory proteins that may also play a role in cell regulation. Two talks focused on the protein p12 I , which is targeted to the endoplasmic reticulum/Golgi of infected cells and decreases MHC class I trafficking to the cell surface. R. Fukomoto from G. Franchini's lab presented data that p12 I decreases activation of the transcription factor NFATI in T cells by binding to LAT and inhibiting T-cell receptor signaling. In contrast, M. Lairmore presented data that p12 I expression in Jurkat cells results in ~20-fold activation of NFAT-dependent gene expression in a calciumdependent manner (Kim and Lairmore). These authors also demonstrated that p12 I acts in the endoplasmic reticulum to activate calcium-mediated T-cell activation during the early stages of infection, apparently through an interaction with calcineurin. These studies suggest a prominent role for p12 I in common T cell activation pathways critical to the establishment of a persistent infection. Another protein, p13 II , which was described by V. Ciminale (Silic-Benussi et al.), is localized to the inner mitochondrial membrane and induces changes in Ca 2+ and K + permeability. In culture, p13 II reverses the morphological transformation of rat embryo fibroblasts expressing c-Myc and Ha-Ras and decreases their ability to form tumors in nude mice. There was speculation that HTLV-I encodes p13 II to counteract the growth-inducing properties of the other viral accessory proteins (such as Tax) that are required for establishment of infection, thereby allowing the virus to persist in infected individuals.
In a theme that echoed in several of the simple retrovirus talks, V. Armbruester from the Mueller-Lantzsch laboratory reported on a novel protein generated by alternative splicing of the envelope gene of the HERV-K endogenous retrovirus. The transcript for this protein, np9, is highly expressed in mammary carcinomas and germ cells, and the gene product binds to the LNX protein, which is a ligand of Numb and targets it for proteasomal degradation. Since LNX/Numb/Notch is a known transformation pathway in tumors, Armbruester speculated that np9 may play a role in tumorigenesis by sequestering LNX, thereby stabilizing Numb.

RNA transcription, processing, export and packaging
This session surveyed new findings on retroviral splicing, nuclear retention and export, translation and packaging. J. Madsen and M. Stoltzfus evaluated the role of an exonic splicing silencer (ESS) on HIV-1 replication in cultured T cells. An HIV-1 mutant with ESS substitutions displays a replication-defective phenotype that correlates with increased viral RNA splicing. This mutant was subjected to long-term passage and the viruses that emerged contained second-site reversions in splice sites flanking the exon containing the mutated ESS. Stoltzfus speculated that strains that do not contain the ESS maintain balanced expression of their viral genome by a novel, unknown mechanism.
A. Lever's lab (Poole et al.) described confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of the interaction between HIV-1 Gag and its cognate genomic RNA. Using biotinylated probes to the fulllength, unspliced RNA, he described an initial perinuclear co-localization between Gag and genomic RNA that subsequently shifted to the plasma membrane. Mutation of the Ψ packaging signal partially disrupted the perinuclear and plasma membrane colocalization.
J. Dudley's lab (J. Mertz et al.) described a new MMTV gene that encodes a viral RNA transport protein generated by alternative splicing of the env gene. The gene was designated rem, (for RNA export protein of MMTV RNA). Its product stimulated nucleocytoplasmic transport of the unspliced MMTV transcript, in a manner similar that of the Rec protein of HERV-K [47,48]. This finding indicates that MMTV encodes at least three accessory genes (encoding dUTPase, superantigen, and Rem) in addition to the standard retroviral genes (gag, pol, and env). The Dudley group suggested that since MMTV exhibits a complex genetic structure it should be reclassified as a complex retrovirus.
K. Boris-Lawrie (Roberts et al.) presented genetic and biochemical data showing that the R-U5 region of SNV RNA adopts a stem-loop structure that stimulates cap-dependent translation. RNA affinity and proteomic analysis showed that RNA helicase A (RHA) bound to wild-type RNA but not to mutants containing substitutions in this structure. RHA interaction with SNV R-U5 stimulated translation of unspliced HIV-1 reporter mRNA. Boris-Lawrie speculated that this could occur by rearrangement of intramolecular RNA interactions that disrupt the packaging signal, thus facilitating mRNA translation.
In vivo, only a fraction of HTLV-infected cells actively expresses viral RNA, leading to speculation that the virus negatively regulates gene expression. P. Green (Younis et al.) described a new role for HTLV-1 and -II accessory proteins p30 and p28, respectively, in negatively regulating virus production during chronic infection. Tax/Rex expression was inhibited upon ectopic expression of p28 by a mechanism that involved nuclear retention of the mRNA. Analysis of RNAs bearing a luciferase reporter gene showed that the 3' splice junction was sufficient to confer this nuclear retention. From this work and the studies from the Franchini and Lairmore labs described above, it is clear that HTLV-1 has evolved to regulate its expression in infected cells, thereby evading immune recognition and promoting viral persistence.

Antivirals
As in previous years, there was a strong emphasis on the development of antiviral agents that interfere with reverse transcription. However, noteworthy progress was also reported in efforts to target a number of additional steps in the replication cycle.
S. Sarafianos reported data from E. Arnold's lab (Himmel et al.) on the structure of HIV-1 RT in a complex with an RNase H inhibitor. Interestingly, the binding site of the compound is quite distant (>40 Angstroms) from the RNase H active site and partially overlaps the NNRTI binding pocket. These results raise the intriguing possibility that compounds could be designed that simultaneously act as RNase H inhibitors and as NNRTIs. M. Miller's lab (Shaw-Reid et al.) performed in vitro assays to examine the effect of RT polymerase inhibitors on RNase H activity. They observed that NNRTIs actually increased RNase H activity; structural studies suggested that this enhancement was due to greater accessibility of the DNA/RNA duplex by RNase H. Although a diketo acid RNase H inhibitor displayed decreased potency in the presence of an NNRTI, the diketo acid and NNRTI synergistically inhibited reverse transcription overall.
C. Wild, E. Freed, and colleagues, and independently C. Aiken's lab, previously reported that a dimethyl succinyl betulinic acid derivative (referred to as PA-457 or DSB) potently blocks HIV-1 infectivity by specifically disrupting the cleavage of the CA precursor (composed of CA and spacer peptide SP1) to mature CA [49,50]. The block to CA-SP1 processing prevents proper core condensation in virions released from PA-457-treated cells. Interestingly, HIV-2 and SIV are insensitive to PA-457. This work was extended by the labs of C. Wild and E. Freed (F. Li et al.) to demonstrate that the determinant of PA-457 activity maps to the N-terminus of SP1. In addition, (Adamson et al.) the passaging of HIV-1 at sub-optimal concentrations of PA-457 led to the appearance of PA-457-resistant vari-ants that contain mutations in the C-terminus of CA or the N-terminus of SP1.
A. Lever's lab (Brown et al.) described their efforts to inhibit HIV-1 replication using oligonucleotides that target the viral genome. The authors observed that oligos targeting the packaging signal (specifically stem-loops 2 and 3) disrupt Gag binding and reduced virus infectivity. T. Murakami and coworkers reported the development of an orally bioavailable compound that binds the HIV-1 chemokine coreceptor CXCR4. In culture, the compound potently inhibits infection by HIV-1 isolates that use CXCR4 as a coreceptor, but, as expected, do not block infection by strains that exclusively use CCR5 as a coreceptor. The compound suppressed HIV-1 infection in the hu-PBL-SCID mouse model. The results of this study suggest that this CXCR4 antagonist could potentially be an effective drug in infected humans.
It has been suggested that most virus originating in the central nervous system (CNS) derives from long-lived cells (e.g., macrophages) that would continue to produce virus for a significant period of time after the initiation of antiretroviral therapy. According to this model, CNSderived virus should decay more slowly following the onset of therapy relative to virus derived from the blood. In the last presentation of the conference, data were presented from R. Swanstrom's lab (Harrington et al.) obtained from a study of HIV-1 population dynamics in cerebrospinal fluid (CSF) immediately following the initiation of antiretroviral therapy. Virus isolates in the CSF apparently derive from both the CNS and the blood plasma. Interestingly, using heteroduplex tracking assays, the authors observed that within the first several days following the initiation of therapy CNS-derived isolates in the CSF decline with similar kinetics to isolates shared with the blood, suggesting that virus from both compartments is produced by cells with a short life span.


extfieldsofstudy: Medicine Biology

text:
Endosulfan's effects: inaccurate data.

We would like to address problems with the article “Effect of Endosulfan on Male Reproductive Development” by Saiyed et al. (2003). Our comments are based on extensive discussions with several experts and decision-making bodies on the subject.

First, Saiyed et al. (2003) described the results from a report first submitted in India in July 2002 by the National Institute of Occupational Health (NIOH 2002). We noted with interest that Saiyed et al. (2003) did not include additional or new data in the article beyond what was presented in the original report (NIOH 2002). Consequently, there was no need for discussing the results again.

The original NIOH report (NIOH 2002) was submitted to an expert panel of Indian scientists appointed by the Government of India. The results of this review were reported to the Indian Central Insecticides Board and Registration Committee in April 2003. The committee was highly critical of the overall conduct of the study and concluded that “there is no link established between use of endosulfan in PCK [Plantation Corporation of Kerala] plantations and health problems reported in Padre village”(Central Insecticides Board and Registration Committee 2003). This was further endorsed at a subsequent meeting of the Registration Committee in September 2003. The Indian scientists who reviewed this report evidently had some major advantages of additional information that were not available to the reviewers of the article by Saiyed et al. (2003). Namely, they had access to all of the data, they could consult the authors, and they clearly comprehended the local situation in the region of India under consideration. Therefore, they found that the NIOH study (NIOH 2002) failed to prove what appeared to be an apparently flawed hypothesis.

Referring to the statement by the Central Insecticides Board and Registration Committee (2003), the Minister for Agriculture reiterated in the Indian parliament that “The [Government] of India also constituted an expert committee and based on its recommendations decided that the use of endosulfan be continued as per provisions of Insecticides Act 1968 as there is no link established between the use of endosulfan in PCK plantations and health problems in Padre village.”

In their article in EHP, Saiyed et al. (2003) emphasized the premise that the geographical area studied was unique, with only a single pesticide used over a long period of time. Unfortunately, this is not correct; local records show that several pesticides were applied in this area. Furthermore, what is also apparent from local records is that several of the same pesticides were also commonly used in the control area. Thus, the scientific value of this study is questionable.

For sexual maturity rating (SMR) and hormone levels, the results presented by Saiyed et al. (2003) displayed a relatively poor correlation with age; this is not enough to clearly describe a positive correlation. There are two additional confounding factors: a) small sample size (small number of subjects and only one blood sample per subject), which was recognized by the authors, and b) Saiyed et al. (2003) did not mention the normal biological ranges for either SMR or hormone levels in such a population. For example, serum hormone levels are highly variable and, without reference to what would be considered a normal range, the authors cannot confidently claim that the apparent changes they described were caused by pesticide exposure. This is evident in the last paragraph of the article, in which Saiyed et al. (2003) concluded that “ long-term follow-up of the children is essential to understand the implications.” As a measure of endosulfan exposure, the authors described serum levels of endosulfan and endosulfan sulphate. There are a number of interesting paradoxes with these results. The first is that endosulfan was not used in the area, as described in the article, for 10 months before the study started. Given the rapid biodegradation and subsequent clearance of endosulfan in this type of environment, it is surprising that endosulfan was discovered in these samples. To further confound this, the levels of endosulfan, again as described by Saiyed et al. (2003), were ≥ 0.03 ppb in water and ≥ 0.3 ppb in pond sediments, well below the levels in the serum samples. Endosulfan is rapidly cleared from the body and does not bioaccumulate; thus, the serum levels are clearly at odds with those found in water samples, including those of the control samples. It should also be added that the water levels of endosulfan described by Saiyed et al. (2003) are well below the maximum recommended by the U.S. Environmental Protection Agency (EPA).

The distance between PCK plantations and Vaninagar school is about 3 km. The amount of endosulfan applied in the PCK plantation block closest to Vaninagar school was about 105 g active ingredient (ai)/acre/year, whereas the permissible application rates authorized by the U.S. EPA are > 1,000 g ai/acre/season. Saiyed et al. (2003) did not include this fact in their article.

In the area described by Saiyed et al. (2003), aerial application was carried out by helicopter at a height of about 10 feet above cashew tree plantations. It is highly unlikely that the sprayed endosulfan would drift away all the way to the Vaninagar school area 3 km away. Also, the interval between annual aerial applications was about 11 months. Endosulfan is likely to undergo significant degradation during this period.

In this area, it is highly unlikely that significant amounts of endosulfan would have translocated with rainwater runoff during monsoon rains, which normally occur 5–6 months after aerial application. Under typical conditions in India, significant degradation of endosulfan would have occurred.

The endosulfan residue levels Saiyed et al. (2003) reported in blood samples were 1,000 times higher than those reported in water samples. Based on the physicochemical properties of endosulfan, such a finding is highly unlikely.

Finally, the reproductive effects of endosulfan reported by Saiyed et al. (2003) are inconclusive.

We are concerned with the comment made by EHP’s Science Editor, Jim Burkhart, in the press release dated 1 December 2003 (EHP 2003):

Decades of spraying this pesticide, and only this pesticide, on the village provided a unique opportunity to analyze its impact. Although the sample size is somewhat limited, the results are quite compelling.

Calling the results “compelling” has caused significant paranoia among users of endosulfan and the general public. A number of pesticides were in use around Padre village for many decades, so it was not correct to state that only one pesticide was used.

In conclusion, we hope we have shown that the article by Saiyed et al. (2003) does not contribute to the knowledge on the behavior of endosulfan or to the research to determine the cause of health problems of the people in Padre village.

In view of the foregoing facts, we believe that EHP should reevaluate their review of the article by Saiyed et al. (2003).

The main objective of the Endosulfan Manufacturers and Formulators Welfare Association in India is disseminating scientific facts concerning the use of endosulfan. Because endosulfan is used in several countries, the association watches for global news and information relevant to endosulfan.
Members of the Endosulfan Manufacturers and Formulators Welfare Association are of the strong opinion that  omitted significant information in their published report "Effect of Endosulfan on Male Reproductive Development." Consequently, to uninitiated readers the article presented an alarming picture of endosulfan, a pesticide registered for use in over 70 countries for more than 40 years.  failed to mention the origin of their study. In fact, their study is an integral part of an epidemiologic study submitted to the government of India in 2003, "Report of the Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District" [National Institute of Occupational Health (NIOH) 2003]. After studying this report and other related studies, an expert group appointed by the government of India categorically concluded in February 2003 that "there is no link established between use of endosulfan in PCK [Plantation Corporation of Kerala] plantations and health problems reported in Padre village" (Central Insecticides Board and Registration Committee 2003). The government of India has since accepted this conclusion.
Epidemiology is a science of proving association and not causation. Therefore, we consider the conclusion of  that their "study results suggest that endosulfan exposure may delay sexual maturity and interfere with hormone synthesis in male children" to be unscientific, uncalled for, and objectionable.  failed to mention the fact that, besides endosulfan, a host of other pesticides were also used in both exposed and control study areas. The comment by EHP's Science Editor, Jim Burkhart, in the press release dated 1 December 2003 (EHP 2003) that "decades of spraying this pesticide [endosulfan], and only this pesticide" is therefore erroneous.
The authors failed to mention the actual quantity of endosulfan aerially applied in the cashew plantation block closest to Vaninagar school (exposed group), which was 105 g active ingredient (ai)/acre/year. This rate should be compared with the permissible seasonal application rates elsewhere, including the United States, where 1,000 g ai/acre/year is often exceeded.  failed to take into consideration the fact that the interval between annual aerial applications was nearly 11 months and that the aerial applications were made 5-6 months ahead of monsoon rains. All this must be weighed against the known half-life of endosulfan in conditions in .
In the proceedings of the expert group, the experts on epidemiology and residue science raised serious objections to the study design, sample selection, and residue analysis adopted by the NIOH (2003).
Endosulfan residue levels reported by  in blood samples are 1,000 times greater than the residue levels reported in water samples by the authors in the original report submitted in India (NIOH 2003). The scientific properties of endosulfan do not support this phenomenon. Also, two studies by Kerala Agricultural University (State of Kerala 2001) that preceded the article by  did not find endosulfan residues in water in Padre village.
These are a few representative omissions and flaws in the article by . Knowing the reputation and professional excellence of EHP, I appreciate the chance to address these problems here.

Endosulfan's Effects: Inaccurate Data
We would like to address problems with the article "Effect of Endosulfan on Male Reproductive Development" by . Our comments are based on extensive discussions with several experts and decision-making bodies on the subject.
First,  described the results from a report first submitted in India in July 2002 by the National Institute of Occupational Health . We noted with interest that  did not include additional or new data in the article beyond what was presented in the original report . Consequently, there was no need for discussing the results again.
The original NIOH report (NIOH 2002) was submitted to an expert panel of Indian scientists appointed by the Government of India. The results of this review were reported to the Indian Central Insecticides Board and Registration Committee in April 2003. The committee was highly critical of the overall conduct of the study and concluded that "there is no link established between use of endosulfan in PCK [Plantation Corporation of Kerala] plantations and health problems reported in Padre village" (Central Insecticides Board and Registration Committee 2003). This was further endorsed at a subsequent meeting of the Registration Committee in September 2003. The Indian scientists who reviewed this report evidently had some major advantages of additional information that were not available to the reviewers of the article by . Namely, they had access to all of the data, they could consult the authors, and they clearly comprehended the local situation in the region of India under consideration. Therefore, they found that the NIOH study  failed to prove what appeared to be an apparently flawed hypothesis.
Referring to the statement by the Central Insecticides Board and Registration Committee (2003), the Minister for Agriculture reiterated in the Indian parliament that "The [Government] of India also constituted an expert committee and based on its recommendations decided that the use of endosulfan be continued as per provisions of Insecticides Act 1968 as there is no link established between the use of endosulfan in PCK plantations and health problems in Padre village." In their article in EHP,  emphasized the premise that the geographical area studied was unique, with only a single pesticide used over a long period of time. Unfortunately, this is not correct; local records show that several pesticides were applied in this area. Furthermore, what is also apparent from local records is that several of the same pesticides were also commonly used in the control area. Thus, the scientific value of this study is questionable.
For sexual maturity rating (SMR) and hormone levels, the results presented by  displayed a relatively poor correlation with age; this is not enough to clearly describe a positive correlation. There are two additional confounding factors: a) small sample size (small number of subjects and only one blood sample per subject), which was recognized by the authors, and b)  did not mention the normal biological ranges for either SMR or hormone levels in such a population. For example, serum hormone levels are highly variable and, without reference to what would be considered a normal range, the authors cannot confidently claim that the apparent changes they described were caused by pesticide exposure. This is evident in the last paragraph of the article, in which  concluded that " long-term follow-up of the children is essential to understand the implications." As a measure of endosulfan exposure, the authors described serum levels of endosulfan and endosulfan sulphate. There are a number of interesting paradoxes with these results. The first is that endosulfan was not used in the area, as described in the article, for 10 months before the study started. Given the rapid biodegradation and subsequent clearance of endosulfan in this type of environment, it is surprising that endosulfan was discovered in these samples. To further confound this, the levels of endosulfan, again as described by , were ≥ 0.03 ppb in water and ≥ 0.3 ppb in pond sediments, well below the levels in the serum samples. Endosulfan is rapidly cleared from the body and does not bioaccumulate; thus, the serum levels are clearly at odds with those found in water samples, including those of the control samples. It should also be added that the water levels of endosulfan described by  are well below the maximum recommended by the U.S. Environmental Protection Agency (EPA).
The distance between PCK plantations and Vaninagar school is about 3 km. The amount of endosulfan applied in the PCK plantation block closest to Vaninagar school was about 105 g active ingredient (ai)/acre/year, whereas the permissible application rates authorized by the U.S. EPA are > 1,000 g ai/acre/season.  did not include this fact in their article.
In the area described by , aerial application was carried out by helicopter at a height of about 10 feet above cashew tree plantations. It is highly unlikely that the sprayed endosulfan would drift away all the way to the Vaninagar school area 3 km away. Also, the interval between annual aerial applications was about 11 months. Endosulfan is likely to undergo significant degradation during this period.
In this area, it is highly unlikely that significant amounts of endosulfan would have translocated with rainwater runoff during monsoon rains, which normally occur 5-6 months after aerial application. Under typical conditions in India, significant degradation of endosulfan would have occurred.
The endosulfan residue levels  reported in blood samples were 1,000 times higher than those reported in water samples. Based on the physicochemical properties of endosulfan, such a finding is highly unlikely.
Finally, the reproductive effects of endosulfan reported by  are inconclusive.
We are concerned with the comment made by EHP's Science Editor, Jim Burkhart, in the press release dated 1 December 2003 (EHP 2003): Decades of spraying this pesticide, and only this pesticide, on the village provided a unique opportunity to analyze its impact. Although the sample size is somewhat limited, the results are quite compelling.
Calling the results "compelling" has caused significant paranoia among users of endosulfan and the general public. A number of pesticides were in use around Padre village for many decades, so it was not correct to state that only one pesticide was used.
In conclusion, we hope we have shown that the article by  does not contribute to the knowledge on the behavior of endosulfan or to the research to determine the cause of health problems of the people in Padre village.
In view of the foregoing facts, we believe that EHP should reevaluate their review of the article by .

Endosulfan's Effects: Saiyed's Response
I would like to respond to the comments of both Abraham and Indulkar about the origin and results of our study .
On the basis of media reports of unusual illnesses in Padre village, we were asked by the National Human Rights Commission to investigate these illnesses and determine if they were linked to endosulfan exposure. These reported illnesses consisted of birth defects, growth-and development-related problems in children, neurologic disorders, epilepsy, allergic disorders, cancers, and high rates of suicide. On the basis of our site visit, study of the topography of the area, the report of the Regional Remote Sensing Service Centre (Nageswara Rao PP, personal communication), and information from the Plantation Corporation of Kerala (PCK), we determined that we had a unique opportunity to study various health effects of longterm exposure to endosulfan on human health.
Our study  had two components: a) an investigation of growthand development-related parameters, such as physical growth (height and weight), skin-fold thickness, IQ, study of behavioral problems and scholastic performance, sexual maturity rating (SMR), and an estimation of sex hormones, in schoolchildren exposed to the aerial spray of endosulfan; and b) an investigation of diseases, such as neurologic and psychiatric disorders, infertility, and allergic disorders, in the adult population, which was carried out through secondary data collection from parents of the children.
In our report submitted to the National Human Rights Commission, [National Institute of Occupational Health (NIOH) 2002], we reported a higher prevalence of birth defects in both male and female children, lower mean SMR and serum testosterone levels in male children of similar age, a higher prevalence of scholastic backwardness, and neurobehavioral problems in children who had been exposed to endosulfan through aerial treatment of fields . We suggested a possible link between endosulfan exposure and these problems, which has been reported in a number of published animal experimental studies, for example, endosulfan-related birth defects (Food Machinery and Chemical Corporation 1980; Environmental Health Perspectives • VOLUME 112 | NUMBER 10 | July 2004 García-Rodríguez et al. 1996;Gupta et al. 1978]; scholastic development (Lakshmana and Raju 1994;Paul et al. 1994); and reproductive effects in males (Dalsenter et al. 1999;Sinha et al. 1997Sinha et al. , 2001. We submitted our report to the Registration Committee at their request. The eight-member expert committee, which included two major stakeholders of endosulfan in the country, was set up by the Registration Committee to examine our report along with the report of the Kerala Agriculture University (KAU) and the Fredrick Institute of Plant Protection and Toxicology (FIPPAT). The committee raised issues, many of which are also mentioned in the letters from Abraham and Indulkar, and remarked that "the findings of the NIOH study are not in conformity with the known and accepted properties, chemistry and toxicology of endosulfan" (Dubey 2003). The committee concluded that the study did not establish a link between endosulfan exposure and the health problems in Padre village. The decision of the committee was not unanimous.
One of the important components of any scientific investigation is dissemination of information to other scientists through publication of the results in a suitable journal; therefore, we submitted a manuscript covering part of the study to EHP. The opinion of the expert committee and their deliberations in no way affected our right of scientific communication. Our study was scientifically planned, designed, and carried out by a team of experts, which included epidemiologists, physicians, pediatricians, medical toxicologists, statisticians, analytical chemists, and biochemists, who have years of experience in conducting such studies and have many publications to their credit. Our article ) was based on a portion of the results of the study in children. Some of the other parameters related to growth and development will be communicated at a later date.
Both Abraham and Indulkar comment that several pesticides were used in the area, so endosulfan cannot be blamed for the reported health problems. Based on the topographic study of the area, we were concerned about what was sprayed on the cashew plantation, which covered a very large area on the hills; we believed that compounds sprayed on the plantation would run off into streams used by residents of the village downhill from the plantation. We asked the PCK, the owners of the cashew plantation, to give us information on all pesticides that were sprayed in the study area. On 20 August 2001, PCK informed us that since 1980 they had aerially sprayed endosulfan (0.1% of 35% emulsifiable concentrate) twice a year almost every year. They did not mention the use of any other pesticides. In their letters, Abraham and Indulkar mention the use of other pesticides in the area; this probably refers to the valley where there are small farms that are owned by the local families. The major crop in this valley and in the control area is areca nut, for which Bordeaux mixture (copper sulfate and lime) is used. The small family farms in the valley and in the control village had a similar crop pattern; therefore, we can assume localized ground use of pesticides. This type of localized pesticide use is unlikely to cause significant widespread exposure. Our conclusions  were based on the comparison of the control and study areas on the basis of aerial exposure; therefore, the use of other pesticides, if similar in both the study and control areas, would not affect our conclusions. The issue of finding endosulfan in serum samples of the control population was adequately addressed in the "Discussion" of our article .
In their letters, Abraham and Indulkar comment that downward movement of the pesticides could start only at the onset of monsoons 5-6 months after exposure, by which time no endosulfan would remain because it biodegrades rapidly. The NIOH and three other agencies carried out endosulfan analyses at varying times (January 2001-August 2001) and reported significant amounts of endosulfan in various environmental media. First, the Centre for Science and Environment, New Delhi (a nongovernment organization), analyzed biological and environmental samples for endosulfan residues in January 2001, 1 month after the last aerial spray of endosulfan carried out on 26 December 2000. The results showed that the concentration of endosulfan in three water samples was 7-51 times higher than the maximum residue limit (MRL) (Joshi 2001). Very high levels of endosulfan were reported in samples of human blood, human milk, vegetables, spices, cow's milk, animal tissues, cashews, cashew leaves, and soil. In one of the soil samples, the concentration of endosulfan was 391 times higher than the MRL.
Second, the KAU, Thrissur District (Kerala), India, studied endosulfan levels in soil (n = 4), plants (n = 5), water (n = 5), and sediment (n = 1) in the pond in the valley on 19 February 2001 using an HPLCspectrophotometric detector technique. They reported endosulfan in soil (3,815 ppb on PCK plantation), 55 ppb in the mid-hills, and 315 ppb in the sediment in the pond (Mathew S, personal communication). They also reported 507-858 ppb endosulfan in cashew leaves. Their report clearly demonstrated downward movement of endosulfan from hills to the pond water ( Figure 1). (Mathew S, personal communication).
Third, FIPPAT, Padappai, India, carried out sampling between March and May 2001 in cashew leaves (n = 28), human blood (n = 112), water (n = 30), and soil (FIPPAT 2001). The authors reported up to 3,430 ppb endosulfan in cashew leaves, 1-11 ppb in soil, and no endosulfan in blood or water samples. On examination of the report (FIPPAT 2001), we noted large peaks of alpha-and beta-endosulfan in some chromatographs of soil, leaf, and blood samples that they had not mentioned. We reported this discrepancy to the expert committee and requested action.
The results of the studies by the Centre for Science and Environment in January 2001 (Joshi 2001 Abraham and Indulkar both noted a discrepancy in our article  between endosulfan levels in serum and water. They both assume that the endosulfan exposure occurred only through water, which is not correct. Endosulfan adhers to the soil particles; runoff water then carries the endosulfan attached to soil particles from leaf surfaces and the ground during the first few rainfalls. Also, winter rains are common in this area of India. This view is largely supported by the KAU study (Mathew S, personal communication), which showed high levels of endosulfan (315 ppb) in pond sediment in the study village. Endosulfan attached to soil particles can enter the body  Indulkar raised several points about the results of our article . He incorrectly stated that we found poor correlation of SMR and hormone levels with age; our correlations are shown in Tables 2 and 3 of our article . He also complained that we had not compared the ranges of SMR and hormone levels in our study groups to normal ranges, but we did not consider it necessary because we were comparing the groups with each other. Also, Indulkar's points about the wide variability of hormone levels and small sample size were discussed and clarified in our article.
Both Abraham and Indulkar stated that endosulfan cannot travel a distance of 3-4 km and that it biodegrades quickly. The long half-life of endosulfan in soil is well known and was mentioned in our "Discussion" . The U.S. Environmental Protection Agency (EPA 2002) reported that Endosulfan is a volatile and persistent cyclodiene pesticide that can migrate over a long distance through various environmental media such as air, water and sediment. Once endosulfan is applied to crops, it can either persist in soil as a sorbed phase or be removed through several physical, chemical and biological processes. Recent studies suggest that secondary emissions of residual endosulfan continue to recycle in the global system while they slowly migrated and are redeposited via wet deposition in the Northern Hemisphere. The occurrence of endosulfan in remote regions like the Great Lakes, the Arctic and the mountainous areas is well documented. Endosulfan can also enter the air as adsorbed phase onto suspended particulate matter, but this process does not appear to be a major contributor to long range transport like volatilization.
The information provided above further supports the comment made by Jim Burkhart, EHP Science Editor, that Decades of spraying this pesticide, and only this pesticide, on the village provided a unique opportunity to analyze its impact. Although the sample size was somewhat limited, the results are quite compelling.
We stand by the conclusions in our article .
The author declares he has no competing financial interests.

Defining Wetlands
I read with interest David J. Tenenbaum's article about constructed wetlands in the January 2004 issue of EHP (Tenenbaum 2004). While I noted his caveat that "their design remains a bit of an uncertain art," I think he should have gone further to ensure that engineers do not immediately embrace constructed wetlands as a panacea for waste treatment and decontamination.
I also find myself increasingly unsettled by articles such as this one as potentially and inadvertently capitalizing on the opinion of the populace that wetlands are good placesa widespread view these days (deservedly so) because of excellent advocacy by government agencies and environmental nongovernment organizations such as Ducks Unlimited.
Although the sewage treatment facilities promoted in the article (Tenenbaum 2004) may resemble wetlands, they are far from the real thing, or at least as far as the term is generally understood. In fact, I noted that later in the article Tenenbaum turned his attention to wetland restoration projects. In my experience, these again generally involve real wetlands, so to speak. Thus our wetland contrivances, enhancements, or other more utilitarian versions are more fairly not referred to as "restoration" for the straightforward reason that the former condition of these sites, the target for restoration, did not include sewage treatment.
The waste treatment benefits from constructed wetlands are clear, and Tenenbaum (2004) is to be commended for pointing this out as well as the uncertain risks. We cross the line, however, when we pitch constructed wetlands without mentioning the caveat that these surrogate ecologic features are intended to become contaminated (per heavy metal sinks, for example) and that this will impair other uses for these facilities, including use as habitat for species that rely on wetlands and cannot discern the real from the fake. Nature's way advises us to be responsible about our waste generation at the source and not to dress up the end-ofpipe solution.
The author declares he has no competing financial interests.

Phthalate Exposure and Early Thelarche
Several years ago Colón et al. (2000) reported higher levels of phthalates, particularly di-(2ethylhexyl) phthalate (DEHP) in serum from 41 girls experiencing premature breast development (thelarche) as compared to 35 agematched controls. These data seem puzzling for at least two reasons. First, in light of the pharmacokinetic properties of phthalates, the reported blood levels are very high when compared to more recent exposure information, and, second, toxicologic evidence shows that phthalates do not act like estrogens under in vivo conditions, nor do they affect female sexual development and maturation in rodents. Despite these concerns, the study by Colón et al. (2000) is one of the few studies that has compared chemical exposures with sexual development and, as a consequence, it is now being cited by authors developing A 542 VOLUME 112 | NUMBER 10 | July 2004 • Environmental Health Perspectives hypotheses for future research programs on children's health (e.g., Chapin et al. 2003).
There have been a number of recent scientific developments that relate to these matters. Thus, it seemed timely to summarize the exposure and toxicologic information that would be relevant in assessing whether lowlevel exposure to phthalates, which might be experienced under typical ambient conditions, could influence human female sexual development. Colón et al. (2000) reported an average DEHP concentration of 450 ppb (nanograms per milliliter) DEHP and 3 ppb monoethylhexyl phthalate (MEHP, the first metabolite of DEHP) in serum samples from the referred cases. In comparison, they found 70 ppb DEHP in controls, whereas MEHP was below levels of detection. In general, the measurement of phthalates in biological fluids and other media can be quite problematic because of the potential for laboratory contamination due to the use of flexible vinyl in laboratory equipment and tubing. Also, the use of flexible vinyl in liners for reagent bottles is common (e.g., Kessler et al. 2001). Much of the historical data on phthalate levels is questionable, and investigators are now measuring phthalate metabolites in biological media as a way of avoiding the sample contamination problems (e.g., Blount et al. 2000aBlount et al. , 2000bKessler et al. 2001). However, taking the data at face value, the results are difficult to rationalize with information on phthalate pharmacokinetics and exposure.
Within the population at large, the principal route of exposure to phthalates is via food (e.g., Clark et al. 2003). Ingested phthalates are converted to the corresponding monoesters before absorption (e.g., Kluwe 1982), and blood levels of phthalate diesters are normally very low. To put this into perspective, a blood level of 450 ppb DEHP is approximately 1.2 µM. In a recent pharmacokinetic study of DEHP in the marmoset (Kessler et al. 2004), oral doses of DEHP at levels of 30 and 500 mg/kg resulted in peak blood levels of DEHP of 0.3 and 1.8 µM. Using a linear allometric extrapolation, an oral dose of approximately 300 mg/kg DEHP would be required to produce a blood level of 450 ppb. In contrast, the average DEHP exposure among U.S. children at ages similar to those examined by Colón et al. (2000) is 2.6 µg/kg/day (McKee et al. 2004; calculated from urinary metabolite data of Brock et al. 2003). Thus, the blood concentration data reported by Colón et al. (2000) are very unusual, and, if correct, imply extraordinary exposures by comparison to those of other children of similar ages.
Further, as indicated above, DEHP is rapidly metabolized to its corresponding monoester, MEHP [mono-(2-ethylhexyl) phthalate]. In a pharmacokinetic study in marmosets (Kessler et al. 2004), oral doses of 30 and 500 mg/kg resulted in peak MEHP concentrations of 8 and 66 µM, approximately 20 times greater than the peak DEHP levels at equivalent doses. In contrast, the average MEHP level of 3 ppb reported by Colón et al. (2000) was more than two orders of magnitude below the reported average DEHP level. The only imaginable situation that might produce such anomalous results is if the blood samples had been taken immediately after direct introduction of DEHP into the blood stream. This is theoretically possible, but the reported levels still seem unlikely. The only way in which significant levels of unmetabolized phthalates may enter the blood stream is by way of medical devices including blood bags, tubing, and other medical equipment [e.g., Food and Drug Administration (FDA) 2001]. DEHP is the primary phthalate plasticizer for such vinyl medical devices. However, as shown in an assessment by the FDA (2001), only critical-care procedures could produce blood levels that begin to approach the levels reported by Colón et al (2000). Further, in blood, DEHP is rapidly metabolized with a biological half-life in humans of < 6 hr (e.g., Peck and Albro 1982). Thus, assuming medical treatment to be the explanation for the high DEHP/MEHP ratio reported by Colón et al. (2000), the measured blood levels imply intensive care procedures performed in the few hours proceeding the blood collection. However, given the circumstances this seems unlikely.
In the study by Colón et al. (2000), the blood samples were taken from children after referral to pediatric clinics, that is, after premature thelarche had been observed. Because phthalates are rapidly metabolized and excreted, the blood level data, if not the result of laboratory contamination, could only have reflected exposures occurring shortly before the blood was drawn. For the hypothesized link between phthalate exposure and early breast development to be supported, one would have to assume that these point estimates were indicative of a pattern of exposure that had persisted for an extended period of time. But, as shown above, the conditions leading to the reported blood levels are so unusual that they cannot reflect an extended exposure profile. Analytical difficulties seem a more likely explanation to reconcile the data reported by Colón et al. (2000) with the extensive body of information on phthalate pharmacokinetics.
The second general reason why an association between phthalate exposure and the early onset of puberty in girls is puzzling and seems unlikely is that it is not supported by the toxicologic data. There is a hypothesis that premature thelarche is a consequence of unintentional exposure to estrogen or estrogen-like substances (e.g., Li et al. 2002;Tiwary 1998). It has been reported that some phthalates, although not DEHP, can bind and activate the estrogen receptor under in vitro conditions (e.g., Jobling et al. 1995;Soto et al. 1995); however, subsequent work has shown that this is an artifact of the testing conditions.
As described above, under in vivo conditions the phthalates are rapidly metabolized to the corresponding monoesters, but these metabolites are not active under in vitro conditions (Brady et al. 1998;Harris et al. 1997;Picard et al. 2001). In rats and mice, phthalates are inactive in uterotrophic assays and do not induce vaginal cornification (Kanno et al. 2003;Zacharewski et al. 1998). Similarly, in longer-term studies phthalates do not influence age at vaginal patency or estrous cyclicity and do not otherwise influence sexual development or behavior in female rats (Moore 2000). Some phthalates produce effects in male rats, apparently as a result of alterations in testosterone synthesis (Parks et al. 2000), but these alterations do not produce estrogenlike effects in female rats (Gray et al. 1999;Moore et al. 2001;Mylchreest et al. 1998Mylchreest et al. , 1999. With respect to female reproductive development, the only effect that has been described in rats relates to histologic changes in the ovary that occur at very high doses . These changes seem to be caused by inhibition of aromatase activity in the granulosa cells. Aromatase inhibition reduces conversion of testosterone to estradiol and produces what is effectively an antiestrogenic effect under these circumstances. However, this seems unlikely to have any public health implications, partly because the doses required in rodents are much higher than those to which humans are exposed and also because the aromatase inhibition is dependent on activation of the peroxisome proliferator-activated receptor α (PPARα). In other situations, there is evidence that PPARαdependent effects exhibit profound species specificity, with rodents being very sensitive and humans being much less sensitive, if not refractory (e.g., Klaunig et al. 2003).
In summary, the purported relationship between phthalate exposure and early thelarche (Colón et al. 2000) seems highly unlikely, in part because the reported exposure levels do not seem plausible given other information on phthalate exposure, and also because phthalates do not influence the timing of female sexual development in laboratory studies. Also, in Figure 3, there should not be a measurement for the year 2010. The authors appologize for the errors.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
On the Tetraploid Origin of the Maize Genome

Data from cytological and genetic mapping studies suggest that maize arose as a tetraploid. Two previous studies investigating the most likely mode of maize origin arrived at different conclusions. Gaut and Doebley [7] proposed a segmental allotetraploid origin of the maize genome and estimated that the two maize progenitors diverged at 20.5 million years ago (mya). In a similar study, using larger data set, Brendel and colleagues (quoted in [8]) suggested a single genome duplication at 16 mya. One of the key components of such analyses is to examine sequence divergence among strictly orthologous genes. In order to identify such genes, Lai and colleagues [10] sequenced five duplicated chromosomal regions from the maize genome and the orthologous counterparts from the sorghum genome. They also identified the orthologous regions in rice. Using positional information of genetic components, they identified 11 orthologous genes across the two duplicated regions of maize, and the sorghum and rice regions. Swigonova et al. [12] analyzed the 11 orthologues, and showed that all five maize chromosomal regions duplicated at the same time, supporting a tetraploid origin of maize, and that the two maize progenitors diverged from each other at about the same time as each of them diverged from sorghum, about 11.9 mya.

It is generally believed that maize, Zea mays L., arose as a tetraploid. The evidence for this argument comes from the finding that the maize genome is largely (up to 72%) duplicated [1][2][3][4][5] and that the maize genome aligns in two chromosomal sets with other grass genomes [6], indicating the presence of two maize subgenomes. In addition, because many maize relatives from the tribe Andropogoneae have five haploid chromosomes in their nuclei, the most parsimonious explanation is that two maize progenitors, both bearing five haploid chromosomes, hybridized to give rise to a tetraploid maize. Despite extensive evidence supporting a tetraploid origin for maize, the exact mode of its origin remains unclear.
The primary study investigating the polyploid history of maize was by Gaut and Doebley [7], who examined the pattern of sequence divergence among 14 pairs of duplicated maize genes (homoeologues). They considered three models of tetraploid formation: autotetraploidy (intraspecific genome duplication), genomic allotetraploidy (interspecific genome hybridization) and segmental allotetraploidy (hybridization of two partially differentiated genomes). According to the mechanism of chromosomal pairing during cell division, they predicted a unimodal distribution of synonymous distances (dS ) between maize duplicated genes for the autotetraploid and genomic allotetraploid models, with the single peak of the distribution referring to the time of switch from tetrasomic to disomic inheritance in the case of autotetraploidy, and to the time of divergence of the two diploid progenitor genomes in the case of genomic allotetraploidy. Bimodal distribution of dS was expected for the segmental allotetraploid model, with one peak referring to the divergence time of the two diploid ancestors (the more diverged part of the ancestral genomes) and another representing the time of switch from tetrasomic to disomic inheritance (onset of divergence of non-diverged part of the ancestral genomes). On the basis of a recovered bimodal distribution of dS between the 14 duplicated maize genes, Gaut and Doebley proposed a segmental allotetraploid origin of maize genome and estimated that the two maize progenitors diverged at ∼20.5 million years ago (mya) and that allotetraploidization occurred at ∼11.4 mya.
Using sequence divergence data from two genes (mdh and waxy), they further suggested that one of the maize progenitors was a closer relative of sorghum than of the other maize progenitor.
However, 6 years later, Volker Brendel and colleagues (Iowa State University, Ames) reanalysed Gaut and Doebley's data, and extended the dataset to many additional putative duplicated maize genes. They found that dS follows a normal distribution, indicating a single genome duplication event estimated to occur at ∼16 mya (quoted in [8,9]). Although they used improved models for dS estimation (codon-based likelihood model), their conclusions are tentative because the orthology of the maize duplicates could not be effectively assessed.
In order to identify genes that are clearly related by descent (orthologous), Lai and colleagues sequenced five duplicated loci ( Figure 1A) from the maize genome, Zea mays L. ssp. mays cv. B73, and the orthologous regions from sorghum, Sorghum bicolor (L.) Moench [10]. Their screening of the bacterial artificial chromosome (BAC) libraries, with probes for the five targeted loci of the five targeted chromosomal regions, resulted in single contiguous series of clones (contigs) for each of the regions in sorghum and in two contigs in each case for maize, indicating that all five regions are unique in sorghum and duplicated in maize [10]. Their finding is in agreement with the results of genetic map comparison studies of grass taxa [6,11], showing that the sorghum genome, although having the same number of chromosomes as maize (N = 10), aligns with the rice genome in a single chromosomal set.
Sequencing of 18 maize BACs and six sorghum BACs and their alignment with orthologous regions from the rice genome yielded more than four million base pairs of genetic information, within which Lai et al. identified 11 genes that were shared by rice and sorghum and were duplicated in homologous regions of maize ( Figure 1B, [10]). Using sequence data for the 11 orthologous genes, Swigonova et al. reexamined the probable origin of the maize genome [12]. Having the data from three grass species, including sorghum as a sister taxon to maize and rice as an outgroup, they performed phylogenetic analyses, including maximum parsimony and maximum likelihood methods. However, the resolution within the recovered gene trees, as indicated by low bootstrap values and short internodes, was confounded by the close relationship of sorghum and maize vs. a large evolutionary distance to the outgroup, rice. Only one of the gene trees, the r1/b1 gene tree, was found to be statistically different from a non-resolved (trichotomous) tree, showing that the b1 locus from maize chromosome 2S is slightly closer to sorghum than it is to the r1 locus from maize chromosome 10L, supporting an allotetraploid origin of maize. However, the internode is very short, and raises the possibility that other factors, such as different expression in different tissues, might have influenced the nucleotide substitution rates in r1/b1 genes, leading to the present pattern of observed distances among the r1/b1 gene orthologues. Nevertheless, the short internode in the r1/b1 gene tree is consistent with the important finding of their study that the two maize progenitor genomes and the sorghum progenitor genome diverged from each other within a very short time period.
To examine the robustness of the results obtained from the phylogenetic analyses, Swigonova et al. performed analogous analyses on the 11 gene orthologues [12]. As in the two previous studies, they investigated sequence divergence among the gene duplicates of maize, but extended the examination to the sorghum gene orthologues. Using a codon-based likelihood model for distance estimation [13] and assuming that rice diverged from the ancestor of maize and sorghum at 50 mya [14], they found that rates of synonymous substitution among the 11 orthologous genes differ by 2.6-fold. In order to account for such a significant difference in rate among genes, they estimated the coalescent times for each pair of maize homoeologues and analysed the coalescent times for each pair of sorghum and maize orthologues. To calculate the coalescent time of two orthologues, they developed formulae that incorporated weighted means to account for variance information in the estimates (Lai et al., unpublished). In contrast to previous studies, they used gene-specific rates of synonymous substitution to estimate the coalescent time of two orthologous sequences. Homogeneity tests [7] on coalescent times of each pair of sorghum and maize orthologues showed a common time interval. However, homogeneity tests performed on the maize duplicated genes indicated that the tbp1/2 gene pair of maize diverged at a different time than all other gene duplicates. Moreover, tbp1/2 also exhibited the smallest variance of synonymous distance and the smallest non-synonymous distance, despite the rather elevated rate of synonymous substitution (11.61 × 10 −9 ). Therefore, they suspected that the maize tbp1/2 gene pair is the product of a gene conversion event between the two homoeologous regions that occurred roughly 4.8 mya. All the other maize duplicated genes diverged at the same time, indicating contemporaneous duplication of the five chromosomal regions and therefore supporting whole genome duplication (tetraploidy). Since the coalescent times of each pair of sorghum and maize orthologues are statistically similar to the coalescent times estimated for each pair of maize homoeologues (except tbp1/2 ), the distance analyses furthermore support the results of phylogenetic analyses that the two progenitors of maize diverged from each other at about the same time as both of them diverged from sorghum, approximately 11.9 mya.
In conclusion, Swigonova et al. [12] showed that the origin of maize might be traced to a more recent time than concluded by Gaut and Doebley [7], approximately 11.9 mya when the two maize progenitors and the sorghum progenitor diverged from each other. The time of the tetraploidization cannot be determined from their data. However, if the tbp1/2 gene pair in maize is indeed the result of a homoeologous conversion, then the tetraploidization must have occurred before 4.8 mya and therefore preceded the major expansion of the maize genome by retrotransposition and gene amplification that has been estimated to occur within the last 5 million years [15].