Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies
Packaging the correct genetic material is a key event in the assembly of an infectious virus . In the case of HIV-1 , both the recognition of the genomic RNA , its targeting to the plasma membrane , and assembly of a membrane-enveloped virus around it is carried out by the viral protein Gag ( Rein et al . , 2011; Sundquist and Kräusslich , 2012; Freed , 2015 ) . The nucleocapsid ( NC ) domain of Gag has two zinc knuckle motifs which together with adjacent residues interact with the viral RNA ( Berkowitz et al . , 1993; Lu et al . , 2011b ) . Mutations in NC that interfere with RNA binding lead to formation of non-infectious viruses ( Aldovini and Young , 1990; Gorelick et al . , 1990 ) . Transcription of the integrated HIV-1 provirus results in both spliced and unspliced transcripts , coding for different viral proteins ( Coffin et al . , 1997 ) . Gag selectively packages unspliced , full-length genomic RNA through interactions with its 5’ untranslated region ( 5’UTR ) , which is different in spliced and unspliced constructs ( Lu et al . , 2011b; Kuzembayeva et al . , 2014 ) . In addition to sequences that mediate the packaging of the genomic RNA , the 5’UTR also has sequence elements related to transcription processivity ( TAR ) , binding of tRNALys-3 as a primer for reverse transcription ( PBS ) , and genome dimerization ( DIS ) ( Johnson and Telesnitsky , 2010; Lu et al . , 2011b; Kuzembayeva et al . , 2014 ) . Single-virion fluorescence assays indicate that >90% of released HIV-1 particles have packaged genomic RNA ( Chen et al . , 2009 ) , and packaging of a dimeric genome appears to be strongly preferred ( Sakuragi et al . , 2003; Chen et al . , 2009; Nikolaitchik et al . , 2013 ) . The packaging of a single dimeric genome is independent of RNA mass , since genomes with altered lengths are always packaged as single dimers ( Nikolaitchik et al . , 2013 ) . Genomic RNAs with sequence differences are efficiently packaged as heterodimers as long as they have identical DIS sequences ( Chen et al . , 2009 ) . The ψ/SL3 stem loop region in the 5’UTR was initially identified as a sequence required for efficient packaging of genomic RNA into HIV-1 virions ( Lever et al . , 1989 ) . Later work characterized a more extended core encapsidation signal ( CES ) which also includes the U5:AUG and PBS stems , the dimerization signal ( DIS ) and the splice donor ( SD ) region ( Heng et al . , 2012 ) . Unpaired and weakly paired guanosines in several regions of the CES , including but not limited to ψ/SL3 were identified as important for packaging ( Abd El-Wahab et al . , 2014; Keane et al . , 2015; Kenyon et al . , 2015 ) . Structures of NC complexed with ψ/SL3 ( De Guzman et al . , 1998 ) and other smaller fragments from the 5’UTR ( Lu et al . , 2011b ) have provided atomic details of how the two zinc knuckles and adjacent motifs of NC interact with RNA . A crystal structure of the DIS:DIS dimerization complex ( Ennifar et al . , 2001 ) along with an NMR structure of the entire CES ( Keane et al . , 2015 ) put these structures in the context of a larger RNA . Thus , we now have a detailed understanding of RNA structural determinants for packaging at the level of individual 5’UTR dimers . A single released HIV-1 virus contains one dimeric genomic RNA ( Chen et al . , 2009 ) and 2400 ± 700 Gag molecules ( Carlson et al . , 2008 ) , indicating that the selective genome packaging may depend on more than just the binary NC-CES interaction . Indeed , cross-linking-immunoprecipitation sequencing ( CLIP-seq ) revealed a strikingly different RNA preference of cytosolic and membrane-bound Gag ( Kutluay et al . , 2014 ) . Cytosolic Gag binds tRNA through its otherwise membrane-binding matrix ( MA ) domain , and associates mainly with cellular RNAs through its NC domain . The minor fraction of cytosolic Gag which binds viral RNA has a strong preference for the 5’UTR but also binds to the Rev-responsive element ( RRE ) , whose main function is thought to be in nuclear export ( Kuzembayeva et al . , 2014 ) . On the other hand , Gag in the membrane fraction has lost its tRNA association and is strongly enriched for viral RNA , interacting with equal probability with all parts of the genomic RNA . Existing structural and biochemical data are not sufficient to explain how a pool of cytosolic Gag , binding mainly cellular RNA , assembles into virus particles which package dimeric viral genomes with 90% probability . Here , we have reconstituted the selective genome packaging of HIV-1 in a minimal system using giant unilamellar vesicles ( GUVs ) , recombinant myristoylated HIV-1 Gag and purified RNAs . Gag assembling on GUV membranes selectively packages the HIV-1 5’UTR at subnanomolar concentrations in the presence of excess competitor RNA . We find that the RNA selectivity of Gag is abolished by mutations that subtly affect its ability to cluster on a membrane . Using SHAPE analysis , we find that membrane recruitment of the 5’UTR by Gag does not lead to drastic changes in its secondary structure , and we further demonstrate that the association of soluble Gag with tRNA confers additional RNA packaging selectivity .
We previously reported an in vitro reconstitution of HIV-1 assembly using recombinant myristoylated Gag protein and PI ( 4 , 5 ) P2-containing giant unilamellar vesicles ( GUVs ) ( Carlson and Hurley , 2012 ) . We showed that GUV-bound Gag clusters mimic the behavior of cellular HIV-1 budding sites in their lipid requirements for assembly , nucleic acid- and ESCRT ( endosomal sorting complex required for transport ) protein binding . We reasoned that this system could also be used to study the RNA selection of an assembling HIV-1 virus . The 5’ untranslated region ( 5’UTR ) and the Rev-responsive element ( RRE ) of the HIV-1 genome ( nucleotides 1–345 and 7259–7612 , of the genomic RNA of HIV-1 strain NL4-3 and ARV-2/SF2 , respectively ) , along with a nominally unstructured 378 nt single-stranded control RNA ( RNA378 ) , were produced by in vitro transcription . The 1–345 construct was previously shown to dimerize even in the absence of Gag ( Heng et al . , 2012 ) . RNAs were labeled with the fluorophore Alexa488 on random guanosines , at measured average labeling efficiencies of ~1 . 1–1 . 2 fluorophores per RNA molecule . 100 nM Gag-ATTO594 was premixed with 0 . 5 nM fluorescent RNA , and added to GUVs which were imaged by confocal microscopy starting 10 min after addition of protein and RNA . Due to the reported Mg2+-dependence of the 5’UTR dimerization ( Wilkinson et al . , 2008 ) , 2 mM MgCl2 was included in the final buffer . The addition of Mg2+ to the imaging buffer made the GUV morphology more variable , but this increased morphological heterogeneity did not hamper the experiments nor the image quantification . In order to quantitate the RNA occupancy of assemblies , ten confocal z-stacks were recorded at random positions on each sample , with each data set containing an average of 50–100 GUVs . Strong RNA fluorescence was consistently observed at Gag clusters on GUV membranes , regardless of which RNA was used ( Figure 1a–c , g ) . This RNA recruitment was dependent on the NC domain of Gag since neither a ΔNC Gag nor Gag treated with the Zn-finger disrupting drug AT-2 ( Rossio et al . , 1998 ) was able to recruit fluorescent RNA to GUV membranes ( Figure 1—figure supplement 1 ) . To study the RNA selectivity of assembling Gag in the reconstituted system , we included a ten fold excess ( 5 nM ) of non-fluorescent RNA378 when premixing RNA and Gag . After assembly on GUVs , the fluorescent 5’UTR was still visible at most Gag clusters in the presence of ten fold excess competitor RNA , whereas RRE was present at fewer Gag clusters and at lower fluorescence intensities , and fluorescent RNA378 was virtually absent ( Figure 1d–f , g ) . Image analysis based on automatic segmentation of Gag clusters and measurement of their average RNA fluorescence confirmed the visual impressions ( Figure 1g ) . 10 . 7554/eLife . 14663 . 003Figure 1 . In vitro reconstitution of selective RNA packaging by HIV-1 Gag . ( a ) Confocal fluorescence micrograph of GUVs containing 5% PI ( 4 , 5 ) P2 . 100 nM HIV-1 Gag-ATTO594 and 0 . 5 nM HIV-1 5’UTR-Alexa488 were premixed and added to the exterior of the GUVs which were imaged 10 min later . Upper panel , membrane in red and Gag in white/cyan . Lower panel , RNA . ( b–c ) As ( a ) but with fluorescent HIV-1 RRE and a 378 nt control ssRNA ( RNA378 ) , respectively . ( d–f ) As ( a–c ) but with 5 nM non-fluorescent RNA378 added together with the fluorescent RNAs . ( a–f ) White arrows mark Gag clusters on GUVs , and their position on the corresponding RNA images . Scale bar for all images , 10 µm . ( g ) Quantitation of fluorescent RNA binding to Gag clusters . Gag clusters on membranes were identified in 10 z-stacks each containing ~10 GUVs , using an unsupervised script which calculated the average RNA fluorescence within the clusters . Gag clusters with an average RNA fluorescence >2 . 0 times that of the surrounding membrane were counted as positive for colocalization . All measurements were conducted on the same batch of GUVs and error bars indicate standard deviation between three repeats on separate GUV preparations . n . s . /* , not significant , and significant , respectively , at p<0 . 05 level by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 00310 . 7554/eLife . 14663 . 004Figure 1—figure supplement 1 . RNA recruitment by clusters of Gag with altered NC domain . ( a ) Confocal micrograph of GUV , 10 min after adding 100 nM ΔNC Gag-ATTO594 and 0 . 5 nM 5’UTR -Alexa488 . Upper panel , membrane in red and Gag in white/cyan . Lower panel , RNA . ( b ) As ( a ) , but with full-length Gag treated for 15 min at 37°C with 100 µM of the Zn-finger disrupting compound AT-2 . ( c ) As ( b ) , but Gag treated with DMSO only as negative control for ( b ) . ( a–c ) White arrows mark Gag clusters on GUVs , and their position on the corresponding RNA images . Scale bar for all images , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 004 To further characterize the sensitivity of the in vitro system , we created a set of altered 5’UTR constructs ( Figure 2a ) . 5’UTR 3way1 has several packaging-critical guanines around the three-way junction 1 in the CES mutated to adenines ( G116A , G318A , G320A , G328A , G329A , G331A , G333A ) . This set of mutations was previously found to reduce packaging in a cellular packaging assay ( Keane et al . , 2015 ) . 5’UTR mono has the GCGCGC sequence in DIS replaced by GAGA , creating a monomeric 5’UTR . 5’UTR 3way1-mono combines the 3way1 and mono mutations . Lastly , 5’UTR env-1 represents the first 345 nt of a spliced viral RNA , with the nucleotides after splice donor site G290 replaced by the nucleotides after splice acceptor site A5 ( Stoltzfus , 2009 ) . In a competition assay similar to the one reported in Figure 1d , 100 nM Gag-ATTO594 was premixed with 0 . 5 nM 5’UTR-Alexa488 and 5 nM of either non-fluorescent 5’UTR construct , and added to GUVs ( Figure 2b–f ) . In the presence of 5 nM 5’UTR very little fluorescent 5’UTR could be seen at Gag clusters on GUVs ( Figure 2b , g ) . However , in the presence of either of the altered constructs , more fluorescent 5’UTR was visible at Gag clusters ( Figure 2c–f , g ) . Thus , the in vitro packaging assay is able to detect the lower affinity of assembling Gag for monomeric , 3way1-mutated , and spliced 5’UTRs . 10 . 7554/eLife . 14663 . 005Figure 2 . Effect of 5’UTR alterations on packaging by HIV-1 Gag . ( a ) Schematic of the HIV-1 5’UTR indicating introduced mutations . Blue indicates the position of the GCGCGC to GAGA mutation in the dimerization motif creating the monomerized construct '5’UTR mono' . Red indicates the position of the splice donor site G290 . Replacement of nucleotides after G290 with the sequence from a spliced RNA created the construct ‘5’UTR env-1’ . Cyan indicates the position of packaging-critical unpaired and weakly paired guanines mutated to adenines in the construct '5’UTR 3way1' . PBS , primer binding site . DIS , dimerization signal . SD , splice donor site . ψ , psi stem loop . The schematic shows one monomer of the dimerizing 5’UTR . ( b–f ) Confocal micrographs of GUVs , 10 min after adding 100 nM Gag-ATTO594 , 0 . 5 nM 5’UTR -Alexa488 , and 5 nM non-fluorescent competitor RNA as indicated . Upper panels , membrane in red and Gag in white/cyan . Lower panel , RNA . White arrows mark Gag clusters on GUVs , and their position on the corresponding RNA images . Scale bar for all images , 10 µm . ( g ) Quantitation of fluorescent RNA binding to Gag clusters , performed as for Figure 1g but using a lower threshold of 1 . 25 for counting Gag clusters as positive for fluorescent RNA . n . s . /* , not significant , and significant , respectively , at p<0 . 05 level by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 005 In summary , a minimal system consisting of purified RNAs , Gag , and PI ( 4 , 5 ) P2-containing GUVs recapitulates the selective RNA packaging of an assembling HIV-1 particle at RNA concentrations far below reported solution affinities of NC for 5’UTR . The 5’UTR is strongly selected for in a competition assay , whereas the RRE is only partially selected for after addition of competitor . 5’UTRs that are either monomerized , have packaging-critical guanines mutated to adenines , or represent a spliced viral RNA are less favored in the competition assay . Given the low concentrations at which this selectivity is observed , below the reported 17 nM affinity constant for 5’UTR-NC association ( Heng et al . , 2012 ) , we turned our attention to potential avidity effects due to Gag multimerization . We next sought to determine whether there was a connection between RNA selectivity by Gag , and its assembly into the membrane-bound hexagonal 'immature' lattice found in viral budding sites and released immature virions ( Ganser-Pornillos et al . , 2012; Sundquist and Kräusslich , 2012; Freed , 2015 ) ( Figure 3a ) . To this end , we produced Gag proteins mutated at symmetry contacts in its lattice-forming CA domain . The W316D , M317D mutation ( "Gag two-fold" ) alters a motif at the two fold symmetry contact in the CA C-terminal domain ( CA-CTD ) , which is described in the literature to be important for virus assembly ( Gamble et al . , 1997; von Schwedler et al . , 2003 ) ( Figure 3a–b ) . When this protein was added to GUVs at 100 nM , it bound in a strikingly different way from wild-type Gag , often resulting in a diffuse Gag fluorescence covering the entire GUV membrane ( Figure 3f , g ) . For comparison , wild type Gag essentially only bound to GUVs in clusters even in the absence of RNA ( Figure 3e , g ) . Based on the structure of the immature CA lattice in intact HIV-1 virions ( Schur et al . , 2015 ) , we further produced a triple mutant ( "Gag three-fold": P170E , A174E , E177R ) designed to alter a three-helix interaction at the three fold interface in CA-NTD ( Figure 3a , c ) . This protein bound GUVs similarly to wild-type Gag , albeit with a moderate increase in the diffuse binding first observed for Gag two-fold ( Figure 3g ) . A Gag protein mutated at the six fold interface in CA-CTD ( Figure 3a , d ) with the single-residue replacement K290A ( "Gag six-fold" ) reported to affect virus assembly ( von Schwedler et al . , 2003; Robinson et al . , 2014 ) , also showed an increase in diffuse Gag binding over wild-type Gag ( Figure 3g ) . Combining the two- , three- , and six-fold mutations in one protein ( "Gag 2- , 3- , 6-fold" ) resulted in an assembly phenotype similar to that of Gag two-fold ( Figure 3g ) . Taken together , mutations in the CA-CTD of either the W316 , M317 motif at the two-fold symmetry contact , or of K290 at the six-fold symmetry contact , both described as leading to virus release defects ( von Schwedler et al . , 2003 ) , affect the ability of Gag to cluster on membranes . 10 . 7554/eLife . 14663 . 006Figure 3 . CA domain mutants affect Gag clustering on membranes . ( a ) Schematic of a hexagonal lattice showing the Gag CA domain at the two- , three- and six fold symmetry contacts , as arranged in the immature Gag lattice in HIV-1 assembly sites and immature virions ( Schur et al . , 2015 ) ( PDB entry 4USN used in panel a–d ) . ( b ) Model of the CA domain at the two fold , with mutated residues in “Gag two-fold” marked in red . ( c–d ) As ( b ) , for Gag mutated at the three- and six-fold symmetry contacts , respectively . ( e ) Confocal micrograph of GUVs , 10 min after adding 100 nM Gag-ATTO594 . Membrane in red and Gag in white/cyan . ( f ) As ( e ) , using Gag-ATTO594 two-fold . GUVs with diffuse Gag binding are denoted with white asterisks . ( e–f ) White arrows mark Gag clusters on GUVs . Scale bar , 10 µm . ( g ) Type of Gag binding to GUVs . For each protein , GUVs were counted in 10 confocal z-stacks , and classified according to having no Gag fluorescence , only clustered Gag fluorescence ( black ) , or a diffuse Gag fluorescence covering the entire membrane ( with or without additional brighter clusters , gray ) . All measurements were conducted on the same preparation of GUVs , with error bars indicating the standard deviation between three repeats conducted on separate GUV preparations . *significant at p<0 . 05 level by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 006 Having characterized the membrane binding of the mutant Gag proteins , we proceeded to study their RNA binding and selectivity . We first compared the membrane binding of Gag proteins in the presence of RNA to Gag proteins alone as characterized above . We observed that the presence of RNA increased the clustering of all Gag proteins , and increased the fraction of GUVs which had bound Gag ( Figure 4a–j , Figure 4—figure supplement 1 ) . First , the capacity of the Gag proteins to recruit fluorescent RNA in the absence of competitor was studied . Each Gag construct was premixed with 0 . 5 nM fluorescent 5’UTR and added to GUVs . Recruitment of fluorescent 5’UTR to the GUV membrane was observed for all Gag constructs ( Figure 4a–e ) , with a quantitation showing a slight decrease in recruitment for the Gag two-fold and Gag two- , three- , six-fold mutants ( Figure 4k ) . However , when a ten fold excess of non-fluorescent competitor RNA ( 5 nM RNA378 ) was added , the Gag proteins behaved differently from each other . Wild type Gag retained as much 5’UTR fluorescence in the presence of competitor RNA as in its absence ( Figure 4f , k ) and the Gag three-fold mutant showed only a minor decrease ( Figure 4h , k ) . On the other hand , GUV-bound clusters of the Gag two-fold , Gag six-fold and Gag two- , three- , six-fold mutants lost most of their 5’UTR fluorescence in the presence of competitor RNA ( Figure 4g , i–k ) . Thus , RNA selectivity in this assay closely correlates with clustered binding of Gag to GUVs in the absence of RNA ( compare Figures 3g and 4k ) . In summary , mutations which cause subtle changes in the ability of Gag to multimerize on a membrane drastically decrease the RNA selectivity of assembled Gag clusters , without greatly affecting their ability to bind RNA non-specifically . 10 . 7554/eLife . 14663 . 007Figure 4 . RNA selectivity of CA domain mutants . ( a ) Confocal micrograph of GUV , 10 min after adding 100 nM Gag-ATTO594 and 0 . 5 nM 5’UTR -Alexa488 . Upper panel , membrane in red and Gag in white/cyan . Lower panel , RNA . ( b–e ) As ( a ) using Gag two-fold , Gag three-fold , Gag six-fold and Gag two- , three- , six-fold , respectively . ( f–j ) As ( a–j ) with the addition of 5 nM non-fluorescent RNA378 . ( a–j ) White arrows mark Gag clusters on GUVs , and their position on the corresponding RNA images . Scale bar for all images , 10 µm . ( k ) Quantitation of RNA binding to Gag clusters , performed as for Figure 1g . n . s . /* , not significant , and significant , respectively , at p<0 . 05 level by Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 00710 . 7554/eLife . 14663 . 008Figure 4—figure supplement 1 . Type of Gag binding to GUVs in the presence of RNA . Statistics of Gag membrane binding was performed as for Figure 3g , using the data presented in Figure 4f–j ( ten z-stacks per condition , in three independent repeats ) . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 008 To further understand how membrane association of Gag affects its interactions with the HIV 5’UTR , we performed selective 2’-hydroxyl acylation analyzed by primer extension ( SHAPE ) analysis ( Merino et al . , 2005 ) on the 5’UTR and determined its SHAPE profiles in the presence and absence of Gag constructs either in solution or in association with GUVs . First , we examined the SHAPE profile of the 5’UTR RNA in solution by itself . The RNA-only SHAPE profile is highly consistent with the recently reported solution structure of the HIV-1 RNA packaging signal ( Keane et al . , 2015 ) , with the tandem three-way junctions exhibiting modest SHAPE reactivities , reflecting the loosely base-paired nature of this region . On the other hand , well based-paired stem regions in the DIS , PBS , U5:AUG , and ψ/SL3 sequences exhibit low or no SHAPE reactivities ( Figure 5a ) . The dimer-promoting DIS loop ( G257-C262 ) had no SHAPE reactivity . This is not per se an unequivocal indication of the 5’ UTR being dimeric since DIS has been reported to partially base-pair with U5 in the monomeric conformation of the 5’UTR ( Lu et al . , 2011a ) . However , the SHAPE reactivity of the SD region G289-G292 ( highlighted with a gray shaded box in Figure 5a ) is consistent with the reported 3D structure of the dimeric conformation ( Keane et al . , 2015 ) , not with the completely unprotected loop proposed for the monomeric 5’UTR ( Lu et al . , 2011a ) . A longer 356 nt 5’UTR able to assume both monomer- and a dimer-favoring conformations ( Heng et al . , 2012 ) had a similar SHAPE reactivity to the 345 nt 5’UTR ( Figure 5—figure supplement 1b ) , indicating that it is present mainly as a dimer under the conditions of the study . We next compared the SHAPE profiles of isolated RNA , and RNA in complexes mimicking steps in the genome packaging . Isolated NC domain was selected to represent the initial interaction of a single Gag molecule with the 5’UTR , ΔMA ( 1–119 ) -Gag for a potential soluble Gag-RNA complex involving CA-mediated Gag-Gag interactions , and Gag clusters on GUVs as a model of viral budding sites on the plasma membrane . In all measurements , protein was kept in excess ( solution protein:RNA ratio 8:1 , GUV protein:RNA ratio 20:1 ) . For a SHAPE analysis on GUV-bound RNA , reactions were set up as for confocal imaging , and GUVs were found to be intact during the treatment ( Figure 5—figure supplement 2 ) . The amount of free RNA in the GUV supernatant was typically 10–20% of the total RNA , dependent on the presence of both GUVs and Gag . The SHAPE profiles for 5’UTR were very similar in the presence of NC , ΔMA-Gag , and when bound to Gag clusters on GUVs ( Figure 5b , Figure 5—figure supplement 1 ) . Upon Gag binding , major SHAPE changes were observed in the following clusters: U117-U122 , G234-A242 , A271-G273 , and A302-G309 ( Figure 5b ) . Among them , the two clusters exhibiting decreased SHAPE reactivity ( G234-C242 and A271-G273 ) surround the two guanine-rich bulges on the DIS stem . This Gag-mediated decrease of RNA flexibility is consistent with , but does not necessarily reflect , direct Gag-RNA contacts ( Kenyon et al . , 2015 ) . These clusters show a more pronounced decrease in SHAPE reactivity than the ψ loop G317-G320 . The other two clusters showing major SHAPE reactivity changes , U117-U122 and A302-G309 , are located around the tandem three-way junctions . Both regions exhibit higher SHAPE reactivities upon Gag binding , indicating that protein interactions lead to rearrangement of the local structure that results in higher RNA flexibility . However , GUV recruitment of the complex did not trigger additional SHAPE changes ( Figure 5c , Figure 5—figure supplement 1 ) , indicating that the overall structure of the 5’UTR is not further altered between the states of initial Gag binding in solution and recruitment to assembled Gag on a membrane . Noteworthy , the SHAPE reactivity of the SD region G289-G292 is similar under all conditions studied ( Figure 5a , c , Figure 5—figure supplement 1 ) , indicating that the 5’UTR is maintained in the dimeric conformation over the assembly process . Taken together , we see no evidence for large changes in secondary structures in the 5’UTR when interacting with either Gag in solution or assembled Gag on a membrane , and it appears dimeric under the conditions studied . 10 . 7554/eLife . 14663 . 009Figure 5 . Probing Gag-induced structural changes on the HIV 5’UTR using SHAPE . ( a ) Secondary structure of the HIV 5’UTR RNA with SHAPE reactivities labeled . The secondary structure is based on the reported NMR structure ( Keane et al . , 2015 ) ( PDB 2N1Q ) . Nucleotides exhibiting high , medium , low , or no SHAPE reactivities are labeled in red , orange , cyan , and black , respectively . SHAPE handles are labeled by gray shadows . The 3D structure colored based on the SHAPE profile using the same color scheme is shown as an insert . U5 , U5 region of long terminal repeat . PBS , primer binding site . DIS , dimerization signal . SD , splice donor site . ψ , psi stem loop . AUG , start codon of gag gene . The nucleotides of SD that would be in a loop in the alternative conformation ( 289–292 ) are underlined with a gray box . The schematic shows one monomer of the dimerizing 5’UTR . ( b ) SHAPE profiles of the 5’UTR either alone ( in gray ) or in complex with the following Gag variants: the NC domain of Gag ( in cyan ) , ΔMA-Gag ( in blue ) , and Gag assembled on GUVs ( in red ) . The regions not showing significant SHAPE changes are masked . ( c ) SHAPE changes between the 5’UTR alone and the 5’UTR bound by the Gag on GUV . Nucleotides exhibiting reduced or increased SHAPE value upon complex formation are labeled in cyan and dark red , respectively . Corresponding nucleotide are also labeled on the NMR 3D structure model using the same color scheme . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 00910 . 7554/eLife . 14663 . 010Figure 5—figure supplement 1 . SHAPE changes for protein addition in solution and a longer 5’UTR RNA . ( a ) Changes in SHAPE reactivity are plotted as in Figure 4c , for addition of ΔMA-Gag and NC , respectively , and between a longer 356 nt 5’UTR and the 345 nt 5’UTR used throughout the study . ( b ) Secondary structure of the 356-nt HIV 5’UTR RNA with SHAPE reactivities labeled as in Figure 4a . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 01010 . 7554/eLife . 14663 . 011Figure 5—figure supplement 2 . Gag binding to GUVs under conditions of the SHAPE analysis . Gag and GUVs were prepared as for SHAPE analysis . In the final step DMSO without SHAPE reagent was added and GUVs were imaged by confocal microscopy . A representative field of view is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 011 The membrane-binding matrix ( MA ) domain of cytosolic Gag has been shown to bind primarily tRNA in cells ( Kutluay et al . , 2014 ) . Several reports have shown that RNA binding to the basic patch on the MA domain of HIV-1 Gag can compete with its ability to bind acidic phospholipids such as PI ( 4 , 5 ) P2 ( Alfadhli et al . , 2009 , 2011; Chukkapalli et al . , 2010 , 2013; Dick et al . , 2013; Dick and Vogt , 2014 ) . We wanted to investigate whether Gag-tRNA interactions also play a role in selective RNA packaging . Premixing 100 nM Gag-ATTO594 with 1 µM yeast tRNA before addition to GUVs led to a virtually complete block in Gag assembly on GUVs ( Figure 6a–b ) . Since the amount of Gag on individual GUVs is more variable than the relative RNA fluorescence at Gag clusters , a membrane flotation assay was devised to quantitate the amount of Gag assembled on membranes . 100 nM Gag-ATTO488 was mixed with 0–20 µM tRNA , and incubated for 10 min with 1 . 0 µm large unilamellar vesicles ( LUVs ) containing 5 mol% PI ( 4 , 5 ) P2 . Vesicles were subjected to flotation on a density step gradient and Gag fluorescence in the top fraction was measured . For the tRNA titration , a 50% inhibition of Gag membrane association was evident at 200 nM tRNA ( Figure 6c ) . At 20 µM tRNA , the amount of Gag found in the vesicle fraction dropped to ~10% of the value seen in the absence of RNA ( Figure 6c ) . Next , long viral or unspecific RNAs at 5 nM concentration were added to Gag with or without tRNA . In addition to the above studied 5’UTR , RRE , and RNA378 , an RNA corresponding to the first 1947 nucleotides of the HIV-1 strain NL4-3 genomic RNA was studied ( HIV1947 ) , along with a single-stranded control RNA of similar length ( RNA2266 ) . In the absence of tRNA , all these RNAs slightly increased the amount of Gag associated with vesicles ( Figure 6d ) . In the presence of 20 µM tRNA however , the 5’UTR and the longer HIV1947 RNA ( which contains the 5’UTR sequence ) enhanced membrane association by Gag more than the other RNAs ( Figure 6e ) . RNA378 had no effect on Gag membrane association in the presence of 20 µM tRNA , whereas the effect of RRE or the longer RNA2266 was slight ( Figure 6e ) . In summary , tRNA at micromolar concentrations potently inhibits membrane association of Gag . Under such conditions , Gag that encounters viral RNA has a higher probability of overcoming that inhibition and associating with membrane . 10 . 7554/eLife . 14663 . 012Figure 6 . Effect of tRNA and viral RNA on Gag membrane association . ( a ) Confocal micrograph of GUVs , 10 min after adding 100 nM Gag-ATTO594 . ( b ) Confocal micrograph of GUVs , 10 min after adding 100 nM Gag-ATTO594 , premixed with 1 µM tRNA . ( a–b ) Membrane in red and Gag in white/cyan . White arrows mark Gag clusters on GUVs . Scale bar , 10 µm . ( c ) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488 , tRNA at 0–20 µm , and 0 . 4 mg/mg 1 . 0 µm LUVs . ( d ) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488 and RNAs at 5 nM . ( e ) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488 , 20 µm tRNA , and other RNAs at 5 nM . ( c–e ) All measurements shown in the same panel were conducted on the same LUV preparation , with error bars indicating the standard deviation between three repeats conducted on separate LUV preparations . n . s . /* , not significant , and significant , respectively , at p<0 . 05 level by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 012
Here , we used biochemical reconstitution methods to show that a minimal system consisting of the HIV-1 Gag protein , the HIV-1 5’UTR , and PI ( 4 , 5 ) P2-containing GUVs is capable of selective RNA packaging in the presence of an excess of non-specific RNA . We find a strong correlation between the ability of Gag to multimerize on a membrane and its selective packaging of viral RNA . Mutations in the lattice-forming CA domain that lessen Gag clustering on membranes ( W316D , M317D and K290A , Figure 3b , d , f , g ) drastically lowered its RNA selectivity ( Figure 4g , i , k ) , without affecting the total capacity of Gag to recruit RNA to a membrane ( Figure 4b , d , k ) . Mutations of these residues in the C-terminal part of CA ( CA-CTD ) have previously been described to lead to virus release defects related to aberrant virus assembly ( Gamble et al . , 1997; von Schwedler et al . , 2003 ) . In line with our findings , it has been reported that the amount of viral RNA coprecipitated with cytoplasmic Gag is reduced five fold when its CA-CTD is deleted , whereas the amount of cellular RNA coprecipitated remains the same ( Kutluay and Bieniasz , 2010 ) . On the other hand , in experiments which did not address selectivity , a ΔCA-CTD Gag was still able to tether genomic RNA to the plasma membrane in cells ( Jouvenet et al . , 2009; Sardo et al . , 2015 ) , which is consistent with our finding that the mutant Gag proteins can still recruit 5’UTR although being less selective in the presence of competitors . Mutations at the three-fold symmetry contact in CA-NTD did not alter Gag clustering on membranes ( Figure 3g ) , concurring with observations that a ΔCA-NTD Gag still supports particle release ( Borsetti et al . , 1998; Accola et al . , 2000 ) . Live-cell fluorescence microscopy has delivered important constraints on the stoichiometry and the location of the RNA selection event . Genomic RNA is present at the onset of HIV-1 assembly at the plasma membrane , at which point the number of Gag molecules was reported to be below the detection limit of ~12 molecules ( Jouvenet et al . , 2009 ) . Cytosolic Gag does not form large oligomers ( Robinson et al . , 2014; Hendrix et al . , 2015 ) and mainly associates with cellular RNAs ( Kutluay et al . , 2014 ) . A small fraction of cytosolic Gag is found to be slowly diffusing and consist of oligomers of up to ~5 molecules ( Hendrix et al . , 2015 ) . In a recent study , TIRF microscopy was used to study the monomer-dimer state of genomic HIV-1 RNAs ( Chen et al . , 2016 ) . It was reported that the large majority of plasma membrane-proximal genomic HIV-1 RNAs are monomeric in the absence of HIV-1 Gag . In cells expressing Gag , monomeric genomic RNAs are seen to coalesce to dimers on the membrane , but were not observed to arrive to the membrane as dimers . That study and our results underscore the importance of membrane-localized Gag assembly events in HIV-1 genome selection and packaging . The SHAPE analysis focused on the secondary structure of the 5’UTR RNA in situations representing different stages of packaging ( Figure 5a , b ) . When comparing SHAPE reactivities of isolated 5’UTR , 5’UTR interacting with NC domain in solution , ΔMA-Gag in solution , or recruited to GUV membranes by clusters of full-length Gag , the changes in SHAPE reactivities were consistent with a conservation of secondary structure between all these states . Based on these results , there is no gross unfolding of the dimeric 5’UTR in the early steps of genomic RNA selection and packaging for HIV-1 . This contrasts with reports that the packaging signal of the related retrovirus Murine Leukemia Virus ( MLV ) is partially unfolded in released immature virions ( Grohman et al . , 2014 ) . The SHAPE reactivity pattern indicated that the 5’UTR was , as expected , dimeric in solution . This was observed both with the shorter 345 nt 5’UTR , which has been reported to be locked in a dimer-favoring conformation , and the longer 356 nt 5’UTR which is also capable of assuming a second conformation ( Lu et al . , 2011a; Heng et al . , 2012 ) ( Figure 5—figure supplement 1 ) . In the light of a recent report that most genomic HIV-1 RNAs in cells are monomeric until interacting with Gag on a membrane ( Chen et al . , 2016 ) , the isolated 5’UTR would thus be representative of the genome after engagement with Gag . It is possible that the RNA chaperone activity of Gag may be necessary to expose the 5’UTR and its DIS in the context of a full-length genome . The strongest decrease in SHAPE reactivity upon Gag interaction was found in the guanosine-rich bulges on the DIS stem ( Figure 5c ) , consistent with a recent study using full-length Gag in solution ( Abd El-Wahab et al . , 2014 ) . The increased SHAPE reactivity in central regions of the tandem three-way junction suggests that Gag engagement , while not leading to a drastic rearrangement of the 5’UTR secondary structure , may lead to alterations its 3D structure . Overall , our results show that RNA conformational changes upon binding to membrane-associated Gag are minor in magnitude and thus suggest that conformation rearrangements are likely to be a minor contributor to selective packaging . Cytosolic RNA has been reported to regulate Gag membrane association , effectively increasing its selectivity for the plasma membrane lipid PI ( 4 , 5 ) P2 ( Alfadhli et al . , 2009 , 2011; Chukkapalli et al . , 2010 , 2013; Dick et al . , 2013; Dick and Vogt , 2014 ) . The RNA species that binds the Gag MA domain in cells was identified by deep sequencing as primarily consisting of tRNA ( Kutluay et al . , 2014 ) . Our reconstituted system allowed us to disentangle the effect that different RNAs have on Gag membrane association ( Figure 6 ) . We show that Gag-tRNA interactions may also serve to increase Gag fidelity for viral RNA . In the absence of tRNA , any long RNA caused a slight increase in Gag membrane association . But in the presence of 20 µM tRNA , 5’UTR and a longer 1947 nt fragment of the HIV-1 genome increased Gag membrane association more than other RNAs . We hypothesize that a viral RNA with several high-affinity binding sites for Gag can recruit several Gag molecules to the membrane if only one of them dissociates from its membrane-occluding tRNA . In summary , our study leads to a model for HIV-1 genome selection where the NC domain of Gag is necessary but not sufficient for the selective packaging of the HIV-1 genomic RNA ( Figure 7 ) . Lattice contacts conferring Gag ability to multimerize on a membrane are crucial for the selective packaging of viral RNA . The 5’UTR is recruited to membranes by multimerized Gag without any gross changes to its secondary structure . Moreover , the association of Gag with tRNA serves to inhibit genome-less virus assembly . 10 . 7554/eLife . 14663 . 013Figure 7 . A model for selective HIV-1 genome packaging . Cytosolic Gag ( light gray triangles ) is inhibited from membrane association by its association with tRNA ( yellow ) . Gag ( triangles and hexagons , colored dark gray where forming specific contacts with the packaging signal ) binding viral genomic RNA ( red; thick portions correspond to the 5’UTR , thin portions to the remainder , not shown to scale ) has a higher probability to overcome this inhibition and associate with the plasma membrane . The selectivity of membrane-bound Gag for the viral genome necessitates CA-CA immature lattice contacts . DOI: http://dx . doi . org/10 . 7554/eLife . 14663 . 013
Full-length , N-terminally myristoylated HIV-1 Gag was purified as previously described ( Carlson and Hurley , 2012 ) . Gag was labelled with ATTO488-maleimide or ATTO594-maleimide ( Sigma-Aldrich , St . Louis , MO ) on an engineered cysteine ( A120C ) in the MA-CA linker region . The resulting fluorophore-labeled protein eluted as a monodisperse peak from the final size exclusion column . ΔNC Gag was cloned from the expression plasmid for full-length Gag by deleting the sequence corresponding to the two Zn fingers of NC ( residues 393–448 of full-length Gag , with the initiator Met being residue 1 ) , and expressed and purified analogously to full-length Gag . The nucleocapsid ( NC ) domain of Gag ( residues 378–432 of full-length Gag ) was expressed in E . coli as a C-terminal , TEV-protease cleavable , maltose-binding protein ( MBP ) - ( His ) 6 fusion protein and purified analogously to full-length Gag , using NiNTA- and Heparin affinity columns followed by TEV protease digestion and size exclusion chromatography on a Superdex 75 16/60 column ( GE Healthcare , Uppsala , Sweden ) . ΔMA Gag ( residues 120–500 ) was based on the same synthetic gene as used for full-length Gag , expressed in E . coli as a TEV-cleavable ( His ) 6-MBP- ΔMA Gag-MBP construct , and purified as full-length Gag . Yeast tRNA was purchased from Invitrogen . All other RNAs were generated by in vitro transcription using T7 polymerase , and purified either by sequential LiCl and ethanol precipitation , or polyacrylamide gel electrophoresis . The 5’UTR and RRE RNAs consisted of nucleotides 1–345 and 7259–7612 of the NL4-3 ( GenBank: AF324493 . 2 ) and ARV-2/SF2 ( GenBank: K02007 . 1 ) unspliced RNAs , respectively ( Heng et al . , 2012; Bai et al . , 2014 ) . RNA378 and RNA2266 were transcribed from the first 378 nucleotides of a synthetic gene encoding human VPS25 ( Im and Hurley , 2008 ) , and the first 2266 nucleotides of the gene encoding H . sapiens AIP1/ALIX ( GenBank: NM_013374 . 5 ) , respectively . The integrity of the RNAs was monitored by denaturing formaldehyde-agarose gel electrophoresis . Fluorophore labeling of RNA was carried out using ULYSIS Alexa Fluor 488 ( Molecular Probes , Waltham , MA ) at 90°C for 10 min in the presence of 3 mM MgCl2 followed by rapid cooling to 4°C to refold the labeled RNAs . Labeled RNAs were buffer-exchanged using Bio-Spin 6 spin columns ( BIO-RAD , Hercules , CA ) , and the final degree of labeling was measured using UV-VIS spectrometry . Lipids were acquired from Avanti polar lipids ( Alabaster , AL ) , except the fluorophore ATTO647-DOPE which was acquired from ATTO-TEC GmbH ( Siegen , Germany ) . GUVs with a molar composition of 69 . 9% palmitoyl-oleoyl-phosphatidylcholine ( POPC ) , 25% cholesterol , 5% brain-extracted phosphatidylinositol-4 , 5-bisphosphate ( PI ( 4 , 5 ) P2 ) and 0 . 1% ATTO647-dioleoyl-phosphatidylethanolamine were electroformed as described previously ( Carlson and Hurley , 2012 ) . For LUVs , lipids with a molar composition of 70% POPC , 25% cholesterol , and 5% PI ( 4 , 5 ) P2 were dried over night in a desiccator and resuspended in LUV buffer ( 20 mM Tris , 150 mM NaCl , pH 7 . 4 ) . They were subjected to ten freeze-thaw cycles on liquid nitrogen , followed by eleven times extrusion through a Nuclepore Track-Etched Membrane with 1 . 0 µm pore size ( Whatman , Little Chalfont , United Kingdom ) . GUVs electroformed in 600 mM sucrose were stored at room temperature and imaged within 24 hr . Protein and RNAs were preincubated for 5 min at room temperature in GUV dilution buffer ( 20 mM Tris , 300 mM NaCl , 4 mM MgCl2 , pH = 7 . 4 ) and then diluted 1:1 with GUVs in a Lab-Tek II chambered coverglass ( Fisher Scientific , Waltham , MA ) . Ten minutes after mixing , the reaction was imaged using an LSM 5 LIVE confocal microscope ( Carl Zeiss , Jena , Germany ) . The membrane fluorophore was imaged using the 635 nm laser and a 650 nm long-pass emission filter , Gag using the 532 nm laser and a 550–615 nm emission filter and RNA using the 488 nm laser and a 500–525 nm emission filter . Membrane , protein , and RNA were imaged using separate tracks , and cross-excitation was found to be minimal with the chosen imaging parameters . On each reaction , ten z-stacks ( each consisting of ten slices with 1 . 0 µm spacing ) were acquired at positions selected solely on the basis of containing GUVs , without observing the fluorescence channels prior to acquisition . All experiments shown in the same figure were done within 24 hr using the same GUV batch for comparability . Each such experiment series was repeated on three separate occasions with different GUV preparations . To Gag-ATTO594 , AT-2 ( aldrithiol-2 , Sigma-Aldrich ) dissolved in DMSO was added to a final concentration of 100 µM , followed by a 15 min incubation at 37°C prior to imaging . Statistics on RNA recruitment to Gag clusters was performed as described previously ( Carlson and Hurley , 2012 ) . Briefly , a custom-written script for MATLAB ( Mathworks , Natick , MA ) used the confocal z-stacks to create a binary mask excluding non-membrane voxels , then identified continuous areas with high Gag fluorescence within the membrane mask . Such an area ( Gag cluster ) was considered positive for RNA recruitment if its average RNA fluorescence was >2 . 0 times higher than the average in the GUV membrane outside Gag puncta , except for Figure 2 where a lower threshold of 1 . 25 was used . To quantitate the Gag binding type ( Figure 2g–h ) , all GUVs with a diameter >5 µm were counted . The GUVs were inspected for Gag binding in the 532 nm fluorescence channel , and those GUVs with Gag clusters but areas completely devoid of detectable Gag fluorescence were counted as 'clustered only' The remaining GUVs either had no detectable Gag binding , or had an even fluorescence covering the entire membrane , sometimes with additional brighter areas . These GUVs were counted as 'diffuse binding' . For SHAPE analysis , SHAPE handles were added to both ends of the 345-nt and 356-nt 5’UTR construct as previously reported ( Bai et al . , 2014 ) . Purified RNA samples were annealed at 0 . 1 mg/ml in a buffer containing 50 mM HEPES-KOH pH 7 . 5 , 200 mM KOAc , and 3 mM MgCl2 by heating at 75°C for 2 min and snap cooling on ice . Before SHAPE reactions , 9 μl of annealed 5’UTR RNA at 0 . 1 mg/ml was mixed with 1 μl of variants of Gag protein to achieve a final RNA:protein stoichiometry of ~1:8 ( 0 . 8 µM:7 . 2 µM ) . Matching protein buffer containing 20 mM Tris , 150 mM NaCl , 0 . 1 mM TCEP , pH = 7 . 4 was used as a control . The resulting mixtures were incubated for 15 min at room temperature . SHAPE probing was performed as previously reported ( Berry et al . , 2011 ) with 1-methyl-7-mitroisatoic anhydride ( 1M7 ) as the 2’ hydroxyl-selective electrophile . For SHAPE analysis of GUV-bound RNA , reactions were set up in Lab-Tek II chambered coverglasses as for confocal imaging to ensure identical mixing kinetics and GUV settling as during imaging . Gag and 5’UTR were premixed with GUV dilution buffer to 150 µl total volume and after 5 min at room temperature were added to 150 µl GUVs in imaging chambers for final concentrations of 100 nM Gag and 5 nM 5’UTR . 10 min after mixing , 200 µl supernatant was gently aspirated . 15 µl 1M7 in DMSO was briefly premixed with 35 µl GUV dilution buffer to avoid exposing GUVs to undiluted DMSO , added to the remaining 100 µl GUV solution in the chamber , and mixed by gentle stirring . Eight chambers were used for each reaction . The control reaction was performed by adding DMSO only . For the test of GUV integrity after this treatment ( Figure 5—figure supplement 2 ) , the control reaction was repeated with fluorescent Gag-ATTO594 and imaged by confocal microscopy as described above 5 min after DMSO addition . To measure the amount of free RNA in the GUV reactions , the supernatant which was removed prior to SHAPE reagent addition was centrifuged for 5 min in a tabletop centrifuge at 21 , 000 ×g to pellet any remaining vesicle-bound RNA . The RNA concentration in the supernatant of this sample was measured using a Qubit fluorimeter ( ThermoFisher Scientific , Waltham , MA ) . Raw traces from fragment analysis was analyzed using ShapeFinder ( Vasa et al . , 2008 ) . Gag and RNA were mixed in a total volume of 60 µl LUV buffer containing 1 . 67 mM MgCl2 and incubated for 5 min at room temperature . 40 µl 1 . 0 mg/ml freshly prepared LUVs were added and the samples were incubated for 10 min at 37°C . The reactions were mixed with OptiPrep solution ( Sigma-Aldrich ) and LUV buffer to 700 µl and 30% ( w/v ) iodixanol . This solution was placed at the bottom of ultracentrifuge tubes and overlayed with 900 µl 18% ( w/v ) iodixanol in LUV buffer and 700 µl LUV buffer . The gradients were subjected to ultracentrifugation in an Sw55 . Ti rotor for 3 hr at 45 , 000 rpm at 4°C . At the end of the run the vesicles fractions were collected from the interface between the 0% and 18% iodixanol fraction , and the amount of Gag associated with the vesicles was measured in a GloMax-Multi microplate reader ( Promega , Fitchburg , WI ) using 490 nm excitation light and a 510–570 nm emission filter . All experiments shown in the same panel were conducted with the same preparation of LUVs and each experiment series was repeated on three separate occasions with different LUV preparations
HIV-1 Gag selects and packages a dimeric , unspliced viral RNA in the context of a large excess of cytosolic human RNAs . As Gag assembles on the plasma membrane , the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism . We used a minimal system consisting of purified RNAs , recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA . Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity . We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA . The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another .
HIV-1 is the virus that causes AIDS – short for acquired immune deficiency syndrome – in humans . When HIV-1 infects a person , it targets cells of the immune system , which normally act to defend the body against infections . As the virus spreads from one immune cell to the next , it weakens the immune system so that individuals become more vulnerable to other illnesses . A cell infected with HIV-1 creates new virus particles at its surface and then releases the particles so that they can infect other cells . HIV-1 viruses encode their genetic information as molecules of ribonucleic acid ( RNA ) . However , the host cell also makes many other RNA molecules that do not contain virus genes so there must be a mechanism in place to ensure that the new virus particles only contain viral RNA . An HIV-1 protein called Gag is responsible for assembling new virus particles and several Gag proteins come together on the cell membrane to form a honeycomb-like structure called the immature lattice . However , it is not clear how Gag is able to select the right RNA molecules . To study how RNA is packaged into new HIV-1 particles , Carlson et al . used artificial versions of the cell membrane , viral RNA and the virus protein Gag to create a simple cell-free system . This system shows that all that is needed for viral RNA to be correctly packaged into new HIV-1 particles is for Gag to be attached to the cell membrane in such a way that the lattice forms correctly . Disturbing the immature lattice by altering the Gag proteins can result in a drastic loss of RNA selectivity . Further experiments show that other molecules in host cells called transfer RNAs enhance the ability of Gag to select the RNAs that encode virus genes . Carlson et al . ’s findings reveal a link between the formation of the Gag lattice and the packaging of virus genes into new virus particles . Drugs that inhibit this process could have the potential to be used as therapies against HIV-1 . A future challenge will be to re-create the entire process of HIV-1 assembly in a cell-free system , which would make it easier to develop new drugs that target the process .