in the 24 years since the discovery of hiv-1 1 , with the impetus to cure aids , there have been an unprecedented number of publications examining retroviral polymorphisms .
these have led to significant advances in understanding mechanisms generating them . in particular , when a provirus replicates along with host cellular dna during mitosis , progeny are extremely homogenous .
a case in point is clonal expansion of htlv-1 2 , resulting in small variability over centuries 2 - 5 .
this can be attributed to the high fidelity of mammalian cellular dna replication , mediated by the 3'-5 ' exonuclease proofreading activity of dna polymerase 6 .
when a provirus replicates exogenously by infection , progeny can be extremely heterogeneous 4 , 7 .
since exogenous retroviral replication involves transcription and reverse transcription , both could contribute towards generating polymorphisms .
therefore , for exogenously replicating retroviruses , such as hiv-1 , polymorphisms are primarily generated during error prone reverse transcription ( fig .
in particular the mis - incorporations produced by rna - templated dna synthesis and dna - templated dna synthesis ( fig .
1 ) , have been proven to be exacerbated by dntp imbalances both in vitro and in vivo 11 - 14 .
proof of the existence of these dntp imbalances in a proportion of retroviral permissive cells 13 has provided a mechanistic explanation for observed retroviral g to a hypermutations in vivo 11 , 13 - 17 .
these mis - incorporations result in polymorphisms , since reverse transcriptase lacks 3'-to-5 ' exonuclease proof - reading activity 18 , provided that the mutations result in replication competent virus that is naturally selected in vivo .
more recently , rna editing , through apobec enzymes , that deaminate cytosine to uracil , have also been proposed as a mechanism for retroviral g to a hypermutation 19 .
in fact , the newest role for the hiv-1 vif protein , is to counter the effect of apobec 20 , 21 .
interestingly , vif may also be exploiting the mutation potential afforded by apobec , in generating more hiv-1 diversity 21 , rather than simply providing immunity from the innate host apobec defense mechanism .
importantly , both templated and non - templated miss - incorporations , as well as  slippage 
therefore , in addition to point mutations , in vivo sampled retroviruses also frequently harbor deletions and insertions . here , among the most probable mechanistic explanations , in addition to , or in combination with the above mentioned non - templated additions ,
is forced copy - choice strand switching during reverse transcription followed by mis - alignment and continued polymerization on the new template 23 - 28 .
in essence this is a forced emulation of the reverse transcription " jumps " depicted in figure 1 29 .
this mechanism reconciles the fact that retroviruses can synthesize cdna with only one rna genomic copy 30 whereas viral particles carry 2 copies .
the second would then serve the failsafe function of providing new template to " jump to " when reverse transcriptase encounters a nick .
in addition , forced copy choice strand - switching provides a probable mechanistic explanation for phylogenetic revelations of inter - subtype recombinant hiv isolates 15 , 31 .
the implication is that chimeric exogenous retroviruses , containing transcribed genomic rna from two different proviruses , are produced from doubly or multiply infected cells .
in fact , recombination has now been shown to be the primary driving force for generating hiv-1 variants 32 , 33 , despite cellular retroviral superinfection resistance mechanisms 34 .
these mechanisms are shared by the larger family of elements capable of retro - transposition 7 , 35 .
however , hiv-1 undergoes such rapid exogenous viral replication and turnover 36 - 38 , that natural selection generates enormously heterogeneous populations termed quasispecies 16 , 39 , or swarms , which have either temporally adapted to cell - specific microenvironments , or exist because their specific polymorphisms are neutral .
the proviral hiv-1 ltr sequences first investigated , were from the initial isolate 40 and rapidly became the prototypical ltr .
these sequences orchestrate the cellular rna polymerase ii transcription machinery , in conjunction with viral proteins , and include a tata - box , 3 sp1 sites , 2 nfkb enhancer elements , an rbeiii site and an upstream usf site ( fig .
importantly , the extensive hiv-1 polymorphism detected in vivo , extends to the ltr ( 15 , 42 and references therein ) .
in particular , a length polymorphism immediately 5 ' of the enhancer region , has been detected , investigated , or referred to by a growing list of publications 42 - 60 .
this polymorphism was initially detected as a 24 base pair duplication by golub , li and volsky , looking for ltr variants ; but no regulatory cis - acting motifs were described within it , despite a correlation with increased transcription 43 . blumberg and co - authors , described a similar duplication , in adult and pediatric patients , while looking for variant nef sequences , but again did not find any motifs that might explain functional significance 44 .
koken and co - authors , were the first to refer to this duplication as the 
ctg-motif , and noted that it encompassed a repeat of the sequence 5'-actgctga-3 ' , and further found that a single  ctg-motif virus , could out - compete an isogenic virus containing the duplication , in co - culture experiments , in 42 days 45 .
these authors also noted similarities between the duplicated region and the recently - described tcf-1 ( hlef ) binding site , and pointed out that hivant 70 , as well as sivcpz , have the duplicated  ctg-motif ( 45 and references therein ) .
these authors also pointed out that nakanishi , masamune and kobayashi , had described the 
in fact , in their paper , nakanishi and co - authors designated this region ( -157 to -121 ) the upstream regulatory element ( ure ) and showed that it bound a transcription factor ( designated ure - binding factors c1 and c2 , by emsa - specific analysis with nuclear extracts ) distinct from ap-1 61 .
furthermore , koken and co - authors 45 noted that zeichner , kim and alwine , had published data suggesting positive regulation in the 
soon afterwards , kim , gonzales , zeichner and alwine , published another paper , showing a more profound effect of mutations in the ure - region (  ctg-motif ) , in the context of chromatin and viral replication , but attributed the effect to mutation of the tcf-1 ( hlef ) binding - site 63 . in another paper by koken and co - authors
ctg-motif duplicated isogenic viruses , are out - competed in cell culture assays by the monomeric isomeric virus 46 .
these authors also showed that a circa 64kd protein , binds with specificity to the 5'-actgctga-3 ' sequence , duplicated in the 
ctg-motif duplications and obtained similar emsa results to those of nakanishi and co - authors , but further reported no correlation between the  ctg-motif duplication and disease stage or viral phenotype 46 , 61 .
so , by 1994 , it was already clear that a cis - element , named ure , enhanced viral replication , contained a putative binding site for tcf-1 ( hlef ) , could specifically bind proteins from nuclear extracts , and was encompassed in a frequent nef / ltr duplication (  ctg-motif duplication )
ctg-motif duplication , in tissue culture assays with isogenic viruses , no dramatic effects were observed .
further reports on this region of the hiv-1 ltr , included a paper from michael and co - authors , that detected the tcf-1 duplication in uncultured samples from patients , and suggested it may mediate a repressive effect 47 , and a paper by ait - khaled , also detecting the tcf-1 duplication , in post - mortem samples 48 .
however , in 1996 , in a cross - sectional analysis of nearly 500 hiv-1 ltrs , we published that the  ctg-motif or ure region duplications , could best be aligned as a single larger polymorphism , which we named the most frequent naturally - occurring length polymorphism ( mfnlp ) of the hiv-1 ltr 42 .
furthermore , we proposed that the mfnlp encompassed a binding site for an unpublished nuclear factor named rbf-2 , instead of tcf-1 42 .
this transcription factor was defined in a subsequent publication 64 , by a tga - specificity for the 5'-actgctga-3 ' sequence , in crude nuclear extracts , and correlated with a region of the hiv-1 ltr required for optimal responsiveness to ras signaling through the mapk pathway , in tissue culture transient transfections .
strikingly , a subsequent publication by linqi zhang and co - authors , reported a tcf-1 duplication in a patient that progressed towards aids , despite the lack of nfb binding 49 .
interestingly , these authors suggested a compensatory role for the tcf-1 duplication , in the face of nfb binding - site abrogation , yet they found very little effect of the duplication in tissue culture 49 .
soon after , kirchhoff and co - authors , reported on the presence of the mfnlp sequence in clinical samples , but did not point out that they duplicated rbeiii sequences , responsive to ras 50 .
similarly , zhang and co - authors , reported on more clinical samples with tcf-1 duplications , for which they found no phenotype , but failed to point out that the mfnlps duplicated rbeiii 51 .
quinones - mateu , reported similar detection of sequence duplications , which we called mfnlps 53 . in 1998 , we further expanded our knowledge about the rbeiii and mfnlp - binding proteins , by showing : ( i ) that hlef ( tcf-1 ) can footprint on the wild type site but not on all mfnlps or rbeiii sites ; ( ii ) that all mfnlps bind rbf-2 ; and ( iii ) that a small repressive effect was mediated through the mfnlp in transient transfections 65 . in 1999 naghavi and co - authors also reported tcf-1 duplications in clinical samples 55 , 66 . at this time , we had already attempted to purify the components of rbf-2 by chromatography , including an oligo - affinity step 67 .
purified rbf-2 included proteins of approximately 120 kd and 45kd , however our repeated attempts to obtain peptide sequences from large scale purifications were inconclusive 67 .
importantly , in the lab of robert roeder , naghavi and i began doing some experiments to confirm her sequencing data that predicted various hiv-1 subtypes would not bind to the nre usf site , since the sequence was not highly conserved , as i had shown for samples from the long - standing vancouver lymphadenopathy study ( vlas ) cohort 42 .
while studying the role of usf-1 in hiv-1 ltr - directed transcription and viral replication , we used a double stranded rbeiii site and a mutant rbeiii site , as a non - specific competitor in an emsa with labeled double stranded e - box targets , encompassing the upstream usf-1 site in the prototypical lav hiv-1 ltr sequence , and nuclear extracts ( nre b usf site , fig 2b , lane1 ) .
surprisingly , we found that at 100x molar excess , rbeiii competed with the band shift , but the rbeiii mutant did not ( fig 2b , lane 9 versus lane 10 ) .
although we at first assumed we had made a mistake , we immediately tested our new hypothesis that rbf2 contains usf-1 , by competing a standard nuclear extract rbf2-rbeiii emsa , with the e - box unlabelled target ( fig .
the hallmark of the rbf2 emsa ( fig 2c , lane 1 ) is competition with 100x wild type rbeiii ( fig .
2c , lane 5 ) in the face of no competition with 100x mutant rbeiii ( fig . 2c ,
the specific rbf2 bands were further tested with the non - specific 100x wtnfb sequences from the prototype lav hiv-1 ltr ( fig .
this important result indicated that our initial observation was true , and suggested that rbf2 contains usf-1 .
we therefore compared the emsa mobility of rusf-1 versus nuclear extracts , with an rbeiii target ( fig .
the standard rbf-2 emsa ( fig . 2d , lanes 1 - 4 , lower arrow ) appears to have a specific band that is lower in mobility than the specific band forming with rusf-1 ( fig .
importantly , although the mobility is slightly different , both bands are specific , since the nuclear extract band can be competed with 100x molar excess of wtrbeiii ( lane 2 ) but not with 100x mut rbeiii ( lane 3 ) and the rusf-1 band can be competed with 100x wtrbeiii ( lane 8) but not 100x mutrbeiii ( lane 9 ) .
additionally , antibodies to rusf-1 appear to supershift both rbf-2 and rusf-1 ( lanes 4 and 10 ) , consistent with usf-1 being in rbf-2 . since our focus is on
the mfnlp and mfnlps encompass rbeiii sequences , the above results strongly suggested that mfnlps would also bind usf-1 .
however , the canonical e - box canntg site , recognized by usf-1 , is not present in rbeiii sites .
indeed target detection analysis ( tda ) , with random targets , using usf-1 , does not select an rbeiii site 68
. however , we noted that in the purified rbf-2 preparations there was a circa 120 kd component 67 .
it was then robert roeder who immediately suggested to me that rbf-2 was comprised of usf and tfii - i . because tfiii and usf-1 bind the initiator / e - box region of the hiv-1 ltr , and rbf-2 had been shown to bind an rbei site ( in addition to the rbeiii site ) that overlaps with the initiator sequences ( fig.2a ) 64 , the hypothesis that rbf-2 was comprised of usf and tfii - i seemed plausible .
in fact , i found this was indeed the case , for at least 2 naturally occurring rbeiii hiv-1 ltr sequences ( from clones pmce36.1 and pmce9.104 , described elsewhere 65 ) , one of which is encompassed in an mfnlp ( pmce9.104 ) ( fig .
2d ) , in addition to the prototype hiv-1 ltr lav sequences ( above ) . importantly , since binding of rusf-1 alone ( fig .
2d , lanes 1 and 2 ) and binding of rtfiii alone ( lanes 5 and 6 ) is significantly weaker than when usf-1 is combined with tfiii ( lanes 3 and 4 ) , it is likely that cooperative binding was occurring .
indeed , this important result suggested that usf-1 and tfiii , when integrated at an rbeiii site , exert a stronger effect than individually , thus potentially explaining why their individual binding to an rbeiii site has not been previously observed .
subsequent to our discovery , we asked the laboratory of ivan sadowski to confirm our results .
martin hirst detected both usf and tfii - i in rbf-2 purifications 67 , confirming our results .
all the above initial findings were excluded from our publication about the nre usf subtype sites 69 , in order to better develop them into a clearer picture of the function of usf / tfii - i at the rbeiii sites , in collaboration with the lab of ivan sadowski ( see below ) 70 .
importantly , jeeninga and co - authors , confirmed in 2000 , that rbeiii sites are absolutely conserved , in ltr subtypes a through g 66 .
a publication in 2000 by gomez - roman and co - authors , detected mfnlp sequences , duplicating rbeiii , in a long term non progressor ( ltnp ) , but no deletions to rbeiii sites were found 56 .
these ltnp are clinically distinct groups with lower viral replication and a slower or non - progressive form of hiv-1 infection ( see below ) . in 2000
, chen and co - authors found that an mfnlp from one of their patient samples , created a new ap-1 binding site , and identified c - fos and junb as binding proteins for their mfnlp 57 .
birch and co - authors described in 2001 , ltnp from the sydney blood bank cohort ( sbbc ) , that are accumulating progressively more ltr / nef deletions , and some of these persons harbor rbeiii duplications in the form of mfnlps 58 .
similarly , another ltnp study appears to have ltr / nef sequences progressively accumulating greater deletions over time 60 .
however it has not been at all clear if the ltr or the nef deletions are responsible for the ltnp phenotype . in 2002 , hiebenthal - millow and kirchhoff , found that in primary t cells , the mfnlp appears not to confer a major effect upon viral replication in culture 59 .
a noteworthy observation about this paper is that these authors actually found enhanced transcriptional activity occurred when both the rbeiii site and mfnlps were deleted , and that the mfnlp slightly reduced transcriptional activity .
perhaps the most important paper about the role of the rbeiii site and the mfnlp is that from chen and co - authors 70 .
this paper expands on our initial discovery that rbf-2 is composed of tfiii and usf , based on the tga specificity of the emsa used by bell and sadowski 64 .
rbf-2 appears to be a heterodimer of usf-1/usf-2 ; and tfii - i appears to load usf-1/ usf-2 onto the rbeiii site .
importantly , this paper shows that rbeiii mutant ltrs are unresponsive to cross - linking of the t cell receptor or stimulation with pma and ionomycin .
thus it appears that the phenotype of the rbeiii site and hence the compensatory mfnlp role , is to provide a target for ras / mapk activation of the hiv-1 ltr , in the context of chromatin 70 , 71 .
importantly , the most recent sbbc ltnp study , indicates that there is a convergence in ltr / nef deletions for these ltnps 72 .
these deletions appear to leave the sequences downstream of the nfb sites intact , yet appear to be abrogating rbeiii sites , since they are deleted , without mfnlps .
importantly , there are publications indicating that nef can activate the mapk pathway 73 and increase t cell activation 74 .
therefore nef could be actually acting through the nef / ltr cis - acting rbeiii site , which is abrogated in the ssbc ltnps 72 .
ltrs with normal rbeiii sites and a normal nef sequence , could contribute to high viral replication and progress to aids ( fig 3a ) .
the presence of mfnlps in the context of normal rbeiii sites would simply provide a failsafe function .
however , ltrs with mutated rbeiii site would require an mfnlp , as a compensatory mechanism ( fig 3 b ) . if however , ltrs receive difficult - to - revert nef deletions , either after an rbeiii mutation ( fig .
3d ) , then the selective pressure to maintain the rbeiii site and the mfnlp disappears .
the result of this model , would explain the convergent deletions observed in the ltr / nef ssbc ltnp study , as well as being consistent with the role for rbf-2 , rbeiii and mfnlps . whether my model about ltnp is true or not , the conserved nature of the rbeiii sites in vivo , makes it an important potential target for delivery of chimeric transcription factor repressors , which could emulate the ltnp phenotype , by blocking activation through this highly conserved site 75 .
this is illustrated in figure 4 , where a chimeric transcription factor encompassing a repressor ( such as mcef75 , 76 ) and a dna binding partner ( rbf-2 ) , might have the potential to both block tat - transactivation and interfere with the function of rbeiii .
( a ) synthesis of minus strong stop dna , begins with t - rna ( clover - leaf structure ) binding to the primer binding site ( pbs ) , followed by rna - templated polymerization of minus strong stop dna ( dashed arrow ) .
rnase h degrades the template rna after polymerization ( arrows ) ; ( b ) template switching to the other rna genome copy or to the same rna , occurs via complementary sequences in the repeat region ( r ) . during the template switch
, slippage can occur ; ( c ) minus strand extension could combine with slippage , templated and non - templated polymerization , resulting in duplication of the ure / rbeiii / partial e - box region ( single white box , in rna u3 region ) , generating a first copy of the mfnlp ( two white boxes in u3 region ) , in the minus dna .
( d ) initial synthesis of plus strand dna begins from the central polypurine tract ( cppt ) and from the 3'-polypurine tract ( 3'-ppt ) , resulting in synthesis of a plus strand mfnlp ( four white boxes ) ; ( e ) annealing of the free pbs , further extension and rearrangements , are followed by nuclear import and integration of the proviral hiv-1 sequences .
( f ) the final integrated proviral sequences contain a 5'-ltr and a 3'-ltr with u3 , r , u5 sequences . in this model , both the 5 ' and 3'-ltr would contain the mfnlp . note the nef orf in the 3'-ltr and the nef / ltr sequences in the 5'-ltr .
integrity of the nef tga stop codon ( actgctga ) is critical for rbf-2 binding to the mfnlp .
( a ) schematic representation of the hiv-1 ltr . some transcription factors landmarks ( nfat , usf-1 , rbf2 , nfkb , sp-1 ) known to interact with target sequences on the hiv-1 ltr are indicated in their relative positions .
the -121 position where the mfnlps occur is indicated , with the mfnlp above , bound by rbf2 at the mfnlp rbeiii site . the -453 and
the + 1 site indicates the start of transcription and the + 98 site , the end of tar sequences .
the dashed circles represent the approximate positions of two nucleosomes ( nuc-0 and nuc-1 ) .
a p32-labelled wt rbeiii ds oligo was used as target in emsa with nuclear extracts from hela - s cells ( ne , lanes 1 - 5 ) .
the target site that the respective competitor ds oligo sequences encompass , are designated with a + sign above each lane , and are listed on the right top of the panel .
( d ) rusf-1 specifically binds to rbeiii , but results in a higher mobility emsa than rbf2 .
a p32-labelled wt rbeiii ds oligo was used as target in emsa with nuclear extracts from hela - s cells ( ne , lanes 1 - 4 ) or with rusf-1 ( lanes 5 - 10 ) .
the target site that the respective competitor ds oligo sequences encompass , are designated with a + sign above each lane , and are listed on the right top of the panel .
( d ) rtfiii and rusf-1 bind cooperatively to mfnlp and wt rbeiii sites from 2 patients . a p32-labelled ds oligonucleotide target , encompassing the rbeiii site of either clone pmce36.1 ( lanes 1 , 3 , 5 ) or pmce9.104 ( lanes 2 , 4 , 6 ) was used as target in emsa with either rusf-1 , tfiii , or both , as indicated with a + sign above each lane , and listed at the right top of the panel .
( b to e ) the nre , rbeiii , usf , nfb sequences and emsa conditions have been previously described 55 , 64 , 65 , 67 , 69 .
mfnlps compensate for rbeiii mutations , by maintaining functional coupling , of nef and rbf-2 . in this schematic representation
four states are considered . in a & b , normal progressive disease with high viral titer
would develop . in c & d , difficult - to - revert - mutations to nef or
the rbeiii site , would result in ltnp , and deletions of both rbeiii ltr sequences and nef sequences .
this is because , in our model , nef can activate the mapk pathway , and exert its effects through rbeiii .
delivery of a repressor with the ability to interfere with tat - transactivation and bind to the rbeiii/ mfnlp is hypothesized .
the rna stem - loop structure termed tar , can form a ternary complex with hiv-1 ta and p - tefb , to hyperphosphorylate the carboxy terminal domain ( ctd ) of rna polymerase ii ( polii ) . because mcef interacts with p - tefb and represses hiv-1 tat - transactivation , if tethered to the rbeiii/ mfnlp site , mcef could interfere with the binding of rbf-2 ( 3 ) and tat transactivation ( 1 and 2 ) .
 although the prototypical hiv-1 ltr sequences were determined 22 years ago from the initial isolate , elucidating which transcription factors are critical to replication in vivo , has been difficult . 
 one approach has been to examine hiv-1 ltrs that have gone through the gamut of in vivo mutation and selection , in search of absolutely conserved sequences . in this vein , 
 rbeiii sequences are virtually 100% conserved in naturally occurring hiv-1 ltrs . 
 this is because when they are mutated , the mfnlp recreates an rbeiii site . 
 here , i enumerate some retroviral mutation mechanisms , which could generate the mfnlp . 
 i then review the literature corresponding to the mfnlp , highlighting the discovery in 1999 , that rbeiii and mfnlp sequences , bind usf and tfii - i cooperatively , within the context of earlier and later work that suggests a role in hiv-1 activation , through t - cell receptor engagement and the mapk cascade . 
 one exception to the nearly absolute conservation of rbeiii , has been a group of long term non progressors ( ltnp ) . 
 these patients harbor deletions to the nef gene . 
 however , the nef gene overlaps with the ltr , and the ltnp deletions abrogate rbeiii , in the absence of an mfnlp . 
 i suggest that the mfnlp retains functional coupling between the mapk - mediated effects of nef and the hiv-1 ltr , through rbeiii . 
 i propose that difficult - to - revert - mutations , to either nef or rbeiii , result in the convergent ltnp nef / ltr deletions recently observed . 
 the potential exploitation of this highly conserved protein - binding site , for chimeric transcription factor repression ( ctfr ) of hiv-1 , functionally striving to emulate the ltnp deletions , is further discussed . 
1. HIV-1 polymorphisms
2. The MFNLP
3. Nef, LTNP and RBF-2
Figures and Tables