Author response: Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies (2016)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.14663.001 eLife digest 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. https://doi.org/10.7554/eLife.14663.002 Introduction 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. Results In vitro reconstitution of selective RNA packaging by membrane-bound HIV-1 Gag 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). Figure 1 with 1 supplement see all Download asset Open asset 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. https://doi.org/10.7554/eLife.14663.003 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. Figure 2 Download asset Open asset 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. https://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. RNA selectivity depends upon intact CA domain lattice contacts 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. Figure 3 Download asset Open asset 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 GUVs with diffuse Gag binding are with White arrows mark Gag clusters on GUVs. Scale 10 µm. (g) of Gag binding to GUVs. For each GUVs were counted in 10 confocal and to Gag only Gag fluorescence or a diffuse Gag fluorescence covering the entire membrane or additional clusters, All measurements were conducted on the same of GUVs, with error bars indicating the standard deviation between three repeats conducted on separate GUV preparations. at p<0.05 level by Student's t-test. characterized the membrane binding of the mutant Gag we to study their RNA binding and selectivity. We first the membrane binding of Gag proteins in the presence of RNA to Gag proteins as characterized We observed that the presence of RNA increased the clustering of all Gag and increased the fraction of GUVs which bound Gag (Figure Figure supplement 1). the of the Gag proteins to recruit fluorescent RNA in the absence of competitor was Gag construct was premixed with 0.5 nM fluorescent 5’UTR and added to GUVs. of fluorescent 5’UTR to the GUV membrane was observed for all Gag constructs (Figure with a showing a in recruitment for the Gag two-fold and Gag two-, three- six-fold mutants (Figure However, when a ten fold excess of non-fluorescent competitor RNA (5 nM was the Gag proteins from each type Gag as 5’UTR fluorescence in the presence of competitor RNA as in its absence (Figure and the Gag mutant showed only a minor (Figure On the other hand, GUV-bound clusters of the Gag Gag six-fold and Gag two-, three-, six-fold mutants lost most of their 5’UTR fluorescence in the presence of competitor RNA (Figure Thus, RNA selectivity in this assay with binding of Gag to GUVs in the absence of RNA Figures and In summary, mutations which changes in the ability of Gag to on a membrane the RNA selectivity of Gag clusters, their ability to RNA Figure with 1 supplement see all Download asset Open asset RNA selectivity of CA domain (a) Confocal micrograph of 10 min after adding 100 nM Gag-ATTO594 and 0.5 nM 5’UTR Upper panel, membrane in red and Gag in white/cyan. Lower panel, RNA. As (a) using Gag Gag Gag six-fold and Gag two-, three-, respectively. As with the addition of 5 nM non-fluorescent White arrows mark Gag clusters on GUVs, and their position on the corresponding RNA images. Scale bar for all images, 10 µm. Quantitation of RNA binding to Gag clusters, performed as for Figure n.s./*, not significant, and significant, respectively, at p<0.05 level by t-test. of interactions between the HIV-1 5’UTR and Gag in solution and on membranes To further how membrane association of Gag its interactions with the we performed selective by primer analysis et al., on the 5’UTR and its SHAPE in the presence and absence of Gag constructs either in solution or in association with GUVs. we the SHAPE of the 5’UTR RNA in solution by The SHAPE is with the reported solution structure of the HIV-1 RNA packaging signal (Keane et al., 2015), with the three-way SHAPE the of this On the other hand, stem regions in the DIS, PBS, and ψ/SL3 sequences low or SHAPE (Figure The DIS loop SHAPE This is not per an of the 5’ dimeric since DIS has reported to partially with in the monomeric of the 5’UTR (Lu et al., However, the SHAPE of the region with a in Figure is with the reported structure of the dimeric (Keane et al., 2015), not with the loop for the monomeric 5’UTR (Lu et al., A nt 5’UTR able to both and a (Heng et al., a similar SHAPE to the 345 nt 5’UTR (Figure supplement indicating that it is present mainly as a the of the We next the SHAPE of RNA, and RNA in in the genome NC domain was selected to represent the interaction of a single Gag with the for a potential soluble complex and Gag clusters on GUVs as a of viral budding sites on the plasma membrane. In all protein was in excess GUV For a SHAPE analysis on GUV-bound RNA, were set as for confocal and GUVs were found to be intact the (Figure supplement The of RNA in the GUV was of the RNA, dependent on the presence of both GUVs and Gag. The SHAPE for 5’UTR were very similar in the presence of and when bound to Gag clusters on GUVs (Figure Figure supplement 1). Gag SHAPE changes were observed in the and (Figure the two clusters SHAPE and the two on the DIS This of RNA is but does not contacts et al., 2015). clusters show a more in SHAPE than the loop The other two clusters showing SHAPE and are around the three-way regions SHAPE upon Gag indicating that protein interactions lead to of the structure that results in RNA However, GUV recruitment of the complex did not additional SHAPE changes (Figure Figure supplement indicating that the structure of the 5’UTR is not further altered between the of Gag binding in solution and recruitment to Gag on a membrane. the SHAPE of the region is similar all (Figure Figure supplement indicating that the 5’UTR is in the dimeric over the assembly process. Taken together, we see for large changes in secondary structures in the 5’UTR when interacting with either Gag in solution or Gag on a membrane, and it appears dimeric the Figure 5 with 2 see all Download asset Open asset structural changes on the 5’UTR using (a) structure of the 5’UTR RNA with SHAPE The secondary structure is based on the reported NMR structure (Keane et al., 2015) (PDB or SHAPE are labeled in and respectively. SHAPE are labeled by The structure based on the SHAPE using the same is shown as an region of long PBS, primer binding site. DIS, dimerization signal. SD, splice donor site. ψ, psi stem loop. of The nucleotides of that would be in a loop in the are with a The schematic shows one monomer of the dimerizing 5’UTR. (b) SHAPE of the 5’UTR either or in complex with the Gag the NC domain of Gag and Gag on GUVs The regions not showing SHAPE changes are SHAPE changes between the 5’UTR and the 5’UTR bound by the Gag on or increased SHAPE upon complex formation are labeled in and respectively. are also labeled on the NMR structure using the same The effects of tRNA and viral RNAs on Gag membrane association The membrane-binding matrix (MA) domain of cytosolic Gag has shown to tRNA in cells (Kutluay et al., 2014). have shown that RNA binding to the on the domain of HIV-1 Gag can with its ability to such as et al., 2011; et al., et al., and 2014). We to whether interactions also a in selective RNA 100 nM Gag-ATTO594 with 1 tRNA addition to GUVs to a virtually in Gag assembly on GUVs (Figure the of Gag on individual GUVs is more than the relative RNA fluorescence at Gag clusters, a membrane assay was to quantitate the of Gag on membranes. 100 nM was with and for 10 min with large unilamellar vesicles containing 5 PI(4,5)P2. were to on a and Gag fluorescence in the fraction was For the tRNA a of Gag membrane association was at nM tRNA (Figure the of Gag found in the fraction to of the seen in the absence of RNA (Figure