To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.
KEYWORDS: HIV-1, antiviral, dominant negative, maturation, nuclear entry
ABSTRACT
Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ∼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions.
IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.
INTRODUCTION
Particle maturation occurs late in the HIV-1 replication cycle and is required for making an infectious virus. Immature HIV-1 particles are composed of Gag polyproteins containing segments corresponding to the matrix (MA), capsid (CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2), and p6 proteins. In cells, Gag is myristoylated at its amino terminus; this modification, together with a nearby highly conserved stretch of positively charged residues in the MA region, promotes its association with the plasma membrane, which is necessary for particle assembly. As the assembling particle begins to bud from the cell membrane, the viral protease is activated and cleaves Gag into its individual protein components. The cleavage sites are processed at different rates, with SP1-NC being cleaved the most rapidly, followed by MA-CA, NC-SP2, SP2-p6, and, finally, CA-SP1 (1–4). The first cleavage event releases NC, resulting in the formation of the condensed viral ribonucleoprotein complex (vRNP) and the stable genomic RNA dimer. Subsequently, cleavage at the MA-CA junction releases CA-SP1, leaving MA attached to the inner face of the viral membrane. CA-SP1 then assembles and is slowly cleaved, resulting in formation of the hexameric capsid lattice. Maturation results in the conversion of immature particles, which contain a spherical lattice of Gag and Gag-Pol polyproteins, into mature particles harboring a conical metastable core. The capsid, a closed conical assembly of CA polymers, encases the nucleic acid and protein components necessary for viral replication: two copies of viral genomic RNA and cognate tRNA, reverse transcriptase (RT), integrase (IN), and nucleocapsid (NC), which collectively comprise the vRNP. The capsid and its contents comprise the viral core (recently reviewed in reference 5).
While the starting and ending products of maturation are hexameric lattices, the structural organizations of the immature and mature lattices are distinct. Each lattice involves contacts between the two independently folded domains of CA: the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD). The immature lattice is an incomplete sphere of radially arranged 8-nm Gag hexamers formed by neighboring CA NTD-NTD interactions and stabilized by CA CTD-CTD dimer interactions (6–8). In contrast, the mature lattice adopts a fullerene cone shape, consisting of ∼250 hexamers and 12 pentamers, which are arranged asymmetrically with 7 on one end and 5 on the other (7, 9–13). Once cleaved from MA, the N-terminal 51 residues of CA fold into a beta-hairpin structure (14). The mature CA lattice is composed of hexamers assembled via CA NTD-NTD (11, 12, 15–17) and NTD-CTD (12, 18, 19) interactions, and adjacent hexamers are stabilized via CTD-CTD interactions at the 3-fold interface (15, 16, 20, 21).
Maturation is a critical step in HIV-1 replication, making it an attractive target for antiviral inhibitor development. The use of protease inhibitors is a highly successful strategy for limiting viral replication (22), as perturbations or interruptions in Gag cleavage result in abortive infection (23). Members of a separate class of maturation inhibitors (MIs), typified by bevirimat, bind to the assembled Gag lattice and inhibit cleavage at the CA-SP1 junction (24–27). Allosteric IN inhibitors (ALLINIs) function relatively late in maturation to decouple the incorporation of the vRNP into the viral capsid shell (28–35). Although both MIs and ALLINIs potently inhibit HIV-1 replication, none of these compounds is yet clinically approved.
The effects of inhibiting cleavage at each site in Gag have been studied in both murine leukemia virus (MLV) (36, 37) and HIV-1 (38–41). Using phenotypic mixing strategies to produce viruses harboring ratios of cleavable and noncleavable Gag proteins, both Lee et al. (39) and Müller et al. (40) reported that incorporation of uncleaved MA-CA protein markedly inhibited HIV-1 infectivity, while Checkley and coworkers (38) reported a similar phenotype when the CA-SP1 junction was blocked. Rulli et al. (37) reported that uncleaved p12-CA in MLV is transdominant due to the absence of an N-terminal proline residue on CA. Lee et al. (39), in contrast, demonstrated that replacement of the N-terminal proline of HIV-1 CA by either Lys or Phe does not result in the potent inhibition of infection, suggesting that the uncleaved MA-CA HIV-1 protein impairs infectivity through a mechanism different from that by which p12-CA impairs infectivity in MLV. Notwithstanding these differences, these studies highlight the importance of proper Gag processing for retroviral replication, since relatively small amounts of uncleaved Gag can dramatically inhibit infection. Lee and coworkers reported that HIV-1 particles containing uncleaved MA-CA protein are impaired for reverse transcription and harbor morphologically aberrant and eccentrically located cores, suggestive of a maturation defect (39). However, the effects of the uncleaved protein on maturation were not analyzed in detail.
In the present study, we sought to further define the mechanism by which uncleaved MA-CA reduces HIV-1 infectivity. Based on the results of the previous study (39), we hypothesized that the coassembly of uncleaved MA-CA and cleaved CA proteins within particles perturbs the intermolecular interfaces in the capsid and that the resulting capsids are intrinsically unstable. We observed evidence for coassembly between uncleaved MA-CA and cleaved CA proteins in virus particles but did not observe detectable perturbation of the CA-CA intermolecular interfaces. Additionally, we observed that the particles contained stable cores and were proficient for reverse transcription. Rather, nuclear entry was predominantly inhibited, and integration targeting was also altered. Finally, the results of genetic analysis suggest that the membrane-binding ability of Gag contributes to the antiviral potency of uncleaved MA-CA.
RESULTS
Confirming the transdominant phenotype of uncleaved MA-CA.
Lee et al. (39) reported that incorporation of a Gag protein containing a substitution preventing cleavage of the MA-CA junction (MA Y132I) profoundly reduces HIV-1 infectivity and yields morphologically eccentric particles. To confirm these results, we transfected HEK293T cells with a wild-type (WT) plasmid together with various quantities of an MA-CA plasmid. The resulting particles, normalized for CA content by a p24 enzyme-linked immunosorbent assay (ELISA), were assayed for infectivity in HeLa TZM-bl cells by quantifying the expression of the luciferase reporter gene that is transactivated in the cells upon expression of the HIV-1 Tat protein from integrated proviruses. Consistent with the findings of the previous study (39), we observed potent inhibition by MA-CA, with 5% MA-CA plasmid resulting in a 70% loss of infectivity and 20% MA-CA plasmid basically ablating HIV-1 infection (Fig. 1A). The cotransfection approach resulted in particles containing both CA and uncleaved MA-CA proteins (Fig. 1B) without affecting particle production (data not shown). We refer to particles generated from WT and MA-CA cotransfections as MA-CA mixed particles.
FIG 1.
Virological properties of HIV-1 containing uncleaved MA-CA. (A) Infectivity of HIV-1 particles produced by transfection of HEK293T cells with a wild-type HIV-1 plasmid together with the indicated fractions of the MA-CA plasmid encoding the Y132I substitution in Gag that prevents cleavage of the MA-CA junction. Shown are the mean values from 5 independent experiments. Error bars represent standard deviations. (B) Immunoblotting analysis of the pelleted particles in which the immunoblots were probed with antiserum specific for CA. Shown are representative results from one of three independent experiments which exhibited similar outcomes. The numbers on the left of the image are molecular masses (in kilodaltons). (C) Particles from MA-CA cotransfections analyzed by thin-section EM. Representative images of mature, immature, eccentric, and empty particle morphologies. (D) Quantitative analysis from thin-section EM. The results are percentages normalized for the different morphologies from the counting of at least 100 particles per experiment; error bars show the standard deviations from two independent experiments.
MA-CA mixed particle morphologies were characterized by thin-section electron microscopy (EM) based on the following classifications: mature, which was characterized by a centrally located electron density often in association with a conical core; immature, which was characterized by a partial or full toroidal electron density beneath the viral membrane; eccentric, which was characterized by a blob-like electron density in association with the viral membrane, most often separated from translucent core-like structures; and empty, which predominantly lacked a clear electron density signal (Fig. 1C). Based on cryogenic electron tomography (cryo-ET) bubblegram imaging of wild-type HIV-1 and morphologically eccentric class II IN mutant particles as well as eccentric particles produced in the presence of ALLINIs, the electron density is coincident with the NC protein and thus maps the position of the vRNP within viral particles (31). Similar to the prior study (39), preparations of MA-CA mixed particles harbored populations of virions with an eccentrically located electron density (Fig. 1D). The percentage of particles with an eccentric particle morphology was proportional to the percentage of MA-CA plasmids in the cotransfections (R2 = 0.90).
Assembly properties of MA-CA mixed particles.
The observed effects of the uncleaved MA-CA protein on the morphology of the viral core suggested that the inhibitory effect may occur through disruption of capsid assembly. Therefore, we sought to determine whether uncleaved MA-CA and cleaved CA proteins coassemble in particles and, if so, what effect that this has on the various intermolecular interfaces necessary for capsid assembly. We employed an approach involving engineered disulfide cross-linking between CA subunits. It has previously been shown that the NTD-NTD intermolecular interface critical for CA hexamer assembly can be covalently stabilized by engineering disulfide cross-links between CA positions A14 and E45, resulting in CA hexamers that can be detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (42). To determine whether the uncleaved MA-CA protein coassembles with CA in virions, we cotransfected a plasmid harboring the A14C substitution with the MA-CA plasmid harboring the E45C substitution (Fig. 2A and B). The predicted molecular weight of the MA-CA/CA heterodimer resulting from disulfide cross-linking is approximately 67 kDa. Under nonreducing conditions, we observed an approximately 70-kDa protein, the intensity of which correlated to the ratios of the two constructs used in the cotransfection. This species was detected by both anti-CA and anti-MA antibodies but was not observed under reducing conditions, indicating that it resulted from disulfide cross-linking and contains the MA-CA protein. We confirmed the results by performing the reciprocal analysis by cotransfecting a plasmid harboring the E45C substitution together with the MA-CA plasmid harboring the A14C substitution. Similar to the original result, we observed the same 70-kDa species under nonreducing conditions (Fig. 2C and D). These results indicate that uncleaved MA-CA is capable of associating with the CA in particles.
FIG 2.
Association of uncleaved MA-CA protein with CA in HIV-1 particles. (A and B) An HIV-1 plasmid encoding the A14C substitution in CA was cotransfected with the MA-CA plasmid encoding the E45C substitution in CA at the indicated percentages. The particles were pelleted, and the proteins were separated by SDS-PAGE under reducing and nonreducing conditions and immunoblotted with antiserum specific for CA (A) and MA (B). (C and D) Results of the same analysis whose results are presented in panels A and B with plasmids encoding the reciprocal Cys substitutions, E45C and MA-CA/CA A14C, for CA (C) and MA (D). Shown are representative results from one of two independent experiments that produced similar outcomes. The numbers on the left of each image are molecular masses (in kilodaltons).
We next asked whether the CA and MA-CA proteins can coassemble in vitro. Using purified recombinant 14C/45C CA and MA-CA proteins, we immunoprecipitated assembly reaction mixtures containing both proteins. Because tubular assemblies of recombinant CA pellet at low speeds, we employed immunoprecipitation by magnetic beads to avoid centrifugation and the consequent pelleting of independently assembled CA tubes. We assembled the CA and MA-CA proteins, either separately or together, in the presence of 1 M NaCl; pelleted them to remove the unassembled proteins; and added the assemblies to anti-MA antibody-coated beads. We observed the coimmunoprecipitation of CA with MA-CA when the proteins were first coassembled in the same assembly reaction (Fig. 3A, lane 4). However, when separately assembled CA and MA-CA proteins were added to the same immunoprecipitation reaction mixture, the quantity of CA in the immunoprecipitate was approximately 20% of that observed with coassembled proteins (Fig. 3A; compare lanes 4 and 5). Our results indicate that CA and MA-CA can coassemble in vitro. We also analyzed the assembled proteins by negative-stain EM. The recombinant 14C/45C CA protein formed long, hollow nanotubes when assembled under high-salt conditions (Fig. 3B, left). Interestingly, when the recombinant CA and MA-CA proteins were coassembled, we did not observe ordered nanotube structures. Rather, we observed densely packed protein aggregates (Fig. 3B, right). Nonreducing SDS-PAGE and Coomassie staining revealed that MA-CA alone did not yield higher-order structures and that CA efficiently formed hexamers in the presence of MA-CA (Fig. 3C). Thus, consistent with our characterizations of virus particles, the recombinant MA-CA protein induced a morphological assembly defect without preventing CA hexamer formation.
FIG 3.
In vitro assembly of recombinant CA and MA-CA proteins. (A) Immunoprecipitation of MA-CA with anti-MA antibody-coated protein A/G magnetic beads. Purified recombinant HIV-1 CA and MA-CA proteins were assembled separately or coassembled (MA-CA Co CA) at 0.8 mg/ml each and pelleted. The assembled proteins were resuspended and captured with magnetic beads coated with MA-specific polyclonal antibody. Immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions and immunoblotted with CA-specific antiserum. Lanes 6 to 8 contain equivalent quantities of the assembled proteins that were added to the beads, analyzed for reference. Shown are representative results from one of three independent experiments which exhibited similar outcomes. (B) Representative negative-stain EM images of recombinant CA (left) and CA coassembled with recombinant MA-CA (right) from one of two independent experiments with similar outcomes. Bars, 500 nm. (C) Assembly reactions from the assay whose results are presented in panel B in which the proteins were separated by nonreducing and reducing SDS-PAGE followed by Coomassie staining. Shown is a representative result from one of two independent experiments with similar outcomes. The numbers on the left of the images in panels A and C are molecular masses (in kilodaltons).
Our results indicate that the uncleaved MA-CA protein induces CA morphological assembly defects both in particles and in in vitro assembly reactions without prohibiting CA hexamer assembly. We next asked whether uncleaved MA-CA perturbs the CA-CA intermolecular interfaces necessary for proper capsid assembly. It has previously been shown that engineered cysteine substitution pairs at the three CA-CA intermolecular interfaces in the viral capsid can yield disulfide cross-links, resulting in CA oligomers that can be detected by SDS-PAGE (90). To test the effects of incorporation of uncleaved MA-CA protein on CA-CA cross-linking at each interface, we cotransfected the MA-CA plasmid with plasmids encoding appropriate Cys-substituted proteins and analyzed the mixed particles by nonreducing SDS-PAGE and immunoblotting. As previously demonstrated, replacement of codons A14 and E45 with Cys resulted in spontaneous disulfide cross-links at the NTD-NTD intrahexameric interface, resulting in a ladder of disulfide-stabilized CA oligomers up to hexamers (42). We observed the efficient formation of these CA forms in particles containing various quantities of uncleaved MA-CA protein (Fig. 4A, lanes 1 to 5). Uncleaved MA-CA inclusion quantitatively reduced the cross-linking to an extent that paralleled what was observed upon cotransfection of the A14C/E45C construct with the wild-type plasmid, consistent with a dilution effect (Fig. 4A, lanes 8 to 12). We attribute the approximately 41-kDa band observed in lane 8 in Fig. 4A to spillover of the sample from lane 7 during loading. In replicates of this experiment, that band was not observed in this sample.
FIG 4.
Incorporation of uncleaved MA-CA protein does not interfere with CA hexamer assembly in virions. HIV-1 plasmids encoding mutations in CA were cotransfected with MA-CA plasmid or wild-type R9 plasmid DNA. Particles were pelleted, and 200 ng p24 of each viral lysate was analyzed by SDS-PAGE under nonreducing conditions and immunoblotted with antiserum specific for CA. (A) A14C/E45C; (B) M68C/E212C; (C) A204C; (D) P207C/T216C. Shown are the results from one of three independent experiments, all of which produced similar outcomes. The numbers on the right of the images in panels A to C and the left of the image in panel D are molecular masses (in kilodaltons).
Similar to the NTD-NTD intrahexameric cross-links formed by the Cys substitutions at CA codons 14 and 45, replacement of M68 and E212 with Cys resulted in intrahexameric cross-links at the NTD-CTD interface (19), also resulting in a ladder of CA oligomers up to hexamers. As was observed for NTD-NTD cross-linking, the pattern was not detectably altered by uncleaved MA-CA incorporation into particles (Fig. 4B; compare lanes 1 to 4 with lanes 8 to 11).
In the HIV-1 capsid, hexamers interact to form a 3-fold interface stabilized by specific amino acid side chains in the C-terminal domains of CA subunits. We probed the formation of the 3-fold interface by two types of engineered disulfide bonds. First, we analyzed cross-linking resulting from a single Cys substitution at position A204 (43), which results in the formation of a CA-CA dimer. As observed in Fig. 4C, formation of the dimer was not detectably affected by the incorporation of uncleaved MA-CA. Second, we observed that the cross-linked CA dimer and trimer species resulting from Cys substitutions at CA positions 207 and 216 (15) were not detectably altered in particles containing uncleaved MA-CA (Fig. 4D, lanes 1 to 3 versus lanes 6 to 8). Collectively, the cross-linking results suggest that MA-CA does not detectably perturb CA-CA intersubunit interfaces during capsid assembly and that the capsids in MA-CA mixed particles, despite their morphologically altered phenotype, assemble into a hexameric lattice.
Uncleaved MA-CA protein associates with stable HIV-1 cores.
Mutations in CA that stabilize or destabilize the viral capsid often result in poorly infectious particles (15, 43–45). Lee et al. (39) previously reported that particles containing uncleaved MA-CA protein are impaired for reverse transcription in target cells. Because mutations that destabilize the HIV-1 capsid often result in reverse transcription defects, we asked whether inclusion of uncleaved MA-CA destabilizes the viral capsid. To test this, we isolated and analyzed cores from particles containing uncleaved MA-CA. Following centrifugation of concentrated particles through a layer of nonionic detergent, density gradient fractions were collected and HIV-1 proteins and viral RNA were analyzed (46). Analysis of the core-associated CA from each fraction by a p24 ELISA revealed elevated levels of CA in MA-CA mixed particles compared with the levels in the wild type (Fig. 5A). However, the anti-CA antibody used in the ELISA can detect both CA and MA-CA proteins, suggesting that the elevated level of core-associated CA detected in mixed particles represents the sum of CA and MA-CA present in purified cores. To determine if uncleaved MA-CA incorporation specifically alters the level of core-associated CA, we concentrated the proteins in the gradient fractions by precipitation with trichloroacetic acid (TCA), separated the proteins by SDS-PAGE, and detected individual viral proteins CA, MA, IN, RT, NC, and, where appropriate, MA-CA by immunoblotting (Fig. 5B). Additionally, we quantified viral RNA by RT quantitative PCR (RT-qPCR) (Fig. 5C). We also quantified the levels of CA species in each fraction of the gradients with a LI-COR Biosciences Odyssey imager and calculated the amount of core-associated CA as a percentage of the entire gradient. The core-associated fractions were identified on the basis of the presence of the known core-associated proteins IN, NC, and RT as well as viral RNA. While there was a small trend toward an elevated level of core-associated CA in MA-CA mixed particles compared with that in the wild type, the difference was not statistically significant (Fig. 5D). Because mutant particles with unstable capsids displayed a distinct reduction in core-associated CA (44), our data suggest that uncleaved MA-CA incorporation does not reduce the intrinsic stability of the capsid. Particles bearing only uncleaved MA-CA exhibited high levels of MA-CA cosedimentation following detergent treatment (Fig. 5B, lower right blot, and Fig. 5D), consistent with a previous report (47). We also observed that uncleaved MA-CA cosedimented with other core-associated components, CA, NC, RT, IN, and viral RNA (Fig. 5B and C), further indicating that MA-CA coassembles with CA in particles. Additionally, we observed a small but statistically significant shift in peak CA density in MA-CA mixed particles, with the peak of cores from particles produced with the 20% MA-CA plasmid being at a density of 1.19 g/ml, which was lower than the density of the control wild-type cores (1.22 g/ml) (Fig. 5E). The density shift was not associated with an obvious change in protein composition or RNA levels (Fig. 5B and C).
FIG 5.
Uncleaved MA-CA protein associates with stable cores. (A) Wild-type control- and MA-CA protein-containing particles were concentrated, and the cores were isolated by sedimentation through a layer of Triton X-100 into a sucrose gradient. Fractions were collected from the top of the gradient (fraction 1) and assayed for the CA concentration by p24 ELISA. The refractive index for each fraction was used to determine the solution density. The results shown are representative of those from six independent experiments. (B) Immunoblotting analysis of fractions from wild-type and MA-CA mixed particles. The proteins present in each fraction were concentrated, analyzed by SDS-PAGE, and immunoblotted with antisera specific for CA, IN, NC, RT, and MA. As a reference, samples (500 ng p24) of the corresponding pelleted virions were analyzed: Lanes *, WT viral lysate; lanes #, MA-CA-containing particles from each corresponding transfection. The lane numbers correspond to the sequential fractions shown in panel A. The immunoblots shown are representative of those from three to six independent experiments. (C) Quantification of HIV-1 RNA in the corresponding fractions. RNA was extracted and quantified by RT-qPCR, and quantification was performed using HIV-1 plasmid DNA as the standard. Shown are the mean values of duplicate measurements from one of two independent experiments, with error bars representing 1 standard deviation. (D) Immunoblot quantification of core-associated CA. Bars represent mean values from three to six independent experiments. Significance was analyzed by an unpaired t test. N.S., not significant; ****, P < 0.0001. (E) Mean density of the peak core-associated CA gradient fraction. The fraction containing the peak core-associated CA was identified from CA band quantification, as described in the legend to panel D. Shown are mean values from 3 to 6 independent determinations, with error bars representing 1 standard deviation. *, P < 0.05 by an unpaired t test.
Although the biochemical analyses suggested that incorporation of uncleaved MA-CA does not result in particles containing intrinsically unstable capsids in vitro, we also sought to determine whether the particles undergo premature uncoating in target cells. To do this, we exploited the well-known property of the host restriction factor TRIMCyp. TRIMCyp, expressed in the cells of some nonhuman primate species, inhibits infection at early postentry stages by binding to the viral capsid (48, 49). Restriction can be saturated by high virus doses (50–53) and can be abrogated by coinoculation with noninfectious particles in trans, so long as the particles are competent for cell entry and contain stable capsids that can be recognized by the restriction factor. Therefore, the ability of a mutant virus to abrogate restriction by TRIMCyp in trans provides a useful assay for capsid stability in target cells (51, 54). To determine if HIV-1 particles containing uncleaved MA-CA protein can abrogate TRIMCyp restriction, we inoculated owl monkey kidney (OMK) cells with titrations of vesicular stomatitis virus G protein (VSV-G)-pseudotyped, mixed viruses together with a fixed, subsaturating quantity of an HIV-green fluorescent protein (GFP) reporter virus and monitored infectivity by analyzing GFP expression by flow cytometry. Although particles composed of 100% MA-CA were unable to overcome restriction, consistent with a previous report (51), we observed that particles containing smaller amounts of uncleaved MA-CA efficiently promoted infection by the reporter virus (Fig. 6A). We did observe a slight reduction in abrogation activity at the highest doses of the inocula, suggestive of a saturation effect.
FIG 6.
Incorporation of uncleaved MA-CA protein does not inhibit the ability of HIV-1 particles to abrogate restriction by TRIMCyp in target cells. (A) Wild-type [pNL4-3ΔE(VSV), where ΔE refers to Env-deficient HIV-1 mutant particles] and MA-CA mixed particles were coinfected with the reporter virus HIV-GFP(VSV) (2 ng of p24) in owl monkey kidney cells. WT and mixed particles were titrated at the amounts shown. Single-cycle infection was monitored by GFP expression, analyzed by flow cytometry. Shown is a representative result from four independent experiments with similar outcomes. Error bars represent standard deviations from technical triplicates. (B) Relative infectivity values of the pseudotyped viruses for which the results are shown in panel A assayed in parallel with nonpseudotyped HIV-1 particles containing the same proportions of uncleaved MA-CA protein. Shown are the mean values from five independent experiments. Error bars represent standard deviations. Significance was analyzed by an unpaired t test. **, P < 0.01; ***, P < 0.001. (C) Immunoblotting analysis of pelleted particles in which the blots were probed with antiserum specific for CA. Samples containing 200 ng of p24 were analyzed. The numbers on the left of the image are molecular masses (in kilodaltons). Shown are representative results from one of three independent experiments which exhibited similar outcomes.
Because VSV-G pseudotyping changes the route of particle entry from fusion to endocytosis (55), we also performed a control experiment to determine if pseudotyping by VSV-G alters the antiviral potency of uncleaved MA-CA incorporation. We observed that uncleaved MA-CA reduced the infectivity of the pseudotyped particles; however, at low MA-CA plasmid doses, the inhibition was approximately 50% less potent than that exhibited by nonpseudotyped HIV-1 particles (Fig. 6B). Immunoblotting of the particles showed comparable levels of MA-CA protein in pseudotyped and control particles, suggesting that the difference in antiviral susceptibility is not a consequence of altered MA-CA incorporation (Fig. 6C). Importantly, the pseudotyped particles produced from 20% MA-CA plasmid cotransfection remained markedly inhibited for infectivity (Fig. 6B).
Collectively, the results from our biochemical and TRIMCyp abrogation assays indicate that HIV-1 particles containing uncleaved MA-CA contain stable capsids. Because TRIMCyp recognizes a hexameric lattice (51, 54), these results further indicate that uncleaved MA-CA does not inhibit hexamer lattice assembly, consistent with our intersubunit cross-linking data (Fig. 4) and in vitro assembly data (Fig. 3C).
Uncleaved MA-CA impairs nuclear entry.
We next sought to identify the precise stage in the viral life cycle at which uncleaved MA-CA impairs infectivity. We started by quantifying virus-cell fusion with the BlaM-Vpr reporter assay. In this approach, reporter viruses were titrated on TZM-bl cells supplemented with CCF4-AM, and fluorescence was measured at 16 h after inoculation. We observed the efficient fusion of viruses produced from MA-CA plasmid cotransfection, including particles containing only MA-CA (Fig. 7A and B). Immunoblotting of pelleted particles demonstrated that the viruses contained similar quantities of the BlaM-Vpr reporter protein (Fig. 7C). We conclude that HIV-1 particles containing uncleaved MA-CA protein are not impaired for fusion with target cells.
FIG 7.
MA-CA mixed particles exhibit impaired nuclear entry. (A) Wild-type and MA-CA-containing particles were assayed for fusion with TZM-bl target cells in the BlaM-Vpr reporter assay. The graph shows representative results from one of three independent experiments. (B) Shown are the mean blue/green fluorescence ratios (from assays employing 10 ng of p24) from three independent experiments. Error bars represent standard deviations. Significance was analyzed by an unpaired t test. N.S., not significant; *, P < 0.05. (C) Immunoblot analysis of pelleted particles for CA and BlaM proteins. WT-, HIV-1 not containing the BlaM-Vpr protein; ΔE, Env-deficient HIV-1 mutant particles. (D) Infectivity, reverse transcription, and nuclear entry of viruses containing uncleaved MA-CA protein. Infectivity was determined by titration on TZM-bl cells and quantification of relative luciferase activity (solid bars). Particles were assayed for synthesis of second-strand transfer DNA (checkered bars) and 2-LTR-circle DNA (open bars) at 8 h and 24 h postinfection of HeLa-CD4+ cells, respectively, by quantitative PCR. Shown are the mean values from four independent experiments, with the copy numbers being normalized to the copy number for the wild type; error bars represent 1 standard deviation. Significance was analyzed by a single-sample t test with a hypothetical mean of 100. N.S., not significant; *, P < 0.05; **, P < 0.01.
We next monitored the effects of MA-CA incorporation on postfusion steps of infection, including reverse transcription and nuclear entry. Lee and coworkers (39) previously observed impaired reverse transcription with particles containing uncleaved MA-CA, suggesting that the inhibitory protein results in an early defect in infection. We performed a similar experiment but employed quantitative PCR to more precisely quantify the levels of HIV-1 DNA produced in cells during infection. To minimize the background PCR signals resulting from the integrated long terminal repeat (LTR) reporter construct present in TZM-bl cells (56), we employed HeLa-CD4+ cells that lack endogenous HIV-1 sequences. In control experiments, we observed that the HeLa-CD4+ cell line exhibited susceptibility to HIV-1 infection comparable to that of TZM-bl cells (data not shown). Following HIV-1 inoculation of the cells, we quantified the formation of late reverse transcription and 2-LTR-circle DNA products (Fig. 7D). We observed no significant reduction in late reverse transcripts in cells infected by particles produced from 5% to 10% MA-CA plasmid cotransfection and a small but statistically significant reduction in cells infected by particles produced from 20% MA-CA plasmid cotransfection. In contrast, particles containing only the uncleaved MA-CA protein were completely inactive for reverse transcription. These results suggest that the major antiviral effect of incorporation of uncleaved MA-CA into HIV-1 particles is manifested at a stage following reverse transcription.
Nonintegrated HIV-1 2-LTR-circle DNA is formed following HIV-1 entry into the cell nucleus; therefore, we quantified 2-LTR circles as an indicator of the efficiency of HIV-1 nuclear entry. We observed significant reductions of 2-LTR-circle DNA in cells infected with MA-CA mixed particles (Fig. 7D). The magnitude of the observed reduction appeared to parallel the infectivity impairment at low levels of MA-CA plasmid cotransfection, with a discrepancy being noted at 20% MA-CA plasmid cotransfection: infectivity was impaired by 99%, while nuclear entry was impaired by only 76%. Therefore, while MA-CA mixed particles exhibit a substantial impairment at nuclear entry, there may be an additional defect at a subsequent step, such as integration.
Uncleaved MA-CA affects integration targeting.
CA and CA-host factor interactions have been implicated in integration efficiency and integration targeting in several HIV-1 studies (57–64). Our original integration site sequencing platform amplified viral-host DNA junctions for Illumina sequencing using primers specific to the viral U5 region (61, 65). To enable site sequencing in cells with preexisting HIV-1 LTR sequences, such as HeLa-P4 cells (66), we modified the U3 region of pNLX.Luc.R-.ΔAvrII (57) to harbor a heterologous 33-bp sequence derived from the U3 region of equine infectious anemia virus (EIAV) 38 nucleotides in from the HIV-1 terminus after reverse transcription (Fig. 8A). The resulting NLX.Luc.R-U3-tag virus supported infection at a level that was virtually indistinguishable from that for the parental NLX.Luc.R- strain (Fig. 8B). The integration sites determined using genomic DNA isolated 5 days after infection, moreover, revealed the expected pattern of HIV-1 integration targeting with respect to several genomic annotations, including transcription units and local gene density surrounding the integration sites (Fig. 8C).
FIG 8.
Description and characterization of U3-tagged virus for integration site sequencing. (A) The upper sequence shows the U3 region of HIV-1NL4-3 that becomes joined to cellular DNA after integration and gap repair; the junction to cell DNA occurs immediately adjacent to the first U3 nucleotide (vertical line). The lower sequence shows the sequence and placement of the heterologous U3 tag (derived from EIAV; in red). (B) HIV-1 infectivities of parental single-round NLX.Luc.R- (WT U3) versus the EIAV-tagged derivative. Two different levels of input virus are shown (average ± standard deviation from two independent experiments). Both viruses at both input levels infected cells similarly (P > 0.05). RLU, relative luminescence units. (C) Integration site distributions of WT versus U3-tagged NLX.Luc.R-. The random integration control (RIC) shows random distributions based on the methods used for DNA library construction.
To test for the possible effects of MA-CA on integration targeting, we cotransfected pNLX.Luc.R-U3-tag with the pNL4-3 MA-CA plasmid and inoculated HeLa-P4 cells with the resulting 5% MA-CA and 20% MA-CA mixed particles. At 4 days postinoculation, cellular DNA was extracted and sheared by restriction endonuclease digestion, and sites of HIV-1 integration were mapped to the human genome. To control for possible dilution effects on the tagged provirus, we performed analyses with viruses produced by cotransfection with parallel quantities of the wild-type (i.e., untagged) pNL4-3 plasmid. While 4,495 unique integration sites were mapped for control viruses that lacked added MA-CA sequences, the lower infectivities of the 5% and 20% MA-CA viruses (Fig. 1A) capped the recovery of the respective integration sites at 1,094 and 205, respectively (Table 1). Whereas 81.3% of WT HIV-1 integrations in this experiment mapped to genes, gene targeting was diminished to ∼77% of integrations for both 5% and 20% MA-CA, which was a significant difference for the 5% MA-CA virus (P = 0.004). The frequencies at which transcriptional start sites (TSSs) and associated CpG islands were targeted did not vary significantly across the different virus preparations. In contrast, targeting of gene-dense regions of chromosomes was significantly affected. While the WT targeted megabase (Mb) regions that harbored, on average, 21.5 genes, MA-CA viruses selected for regions that harbored, on average, 18.6 genes (P = 3.6 × 10−12 and 1.7 × 10−4 for the 5% and 20% MA-CA viruses, respectively). These changes were associated with upticks in targeting of heterochromatic lamina-associated domains (LADs) from 17.2% for the WT to 22.9% (P = 1.9 × 10−5) and 22.4% (P = 0.055) for the 5% and 20% MA-CA viruses, respectively (Table 1). As expected, we did not observe significant changes in integration targeting in cells infected with the corresponding viruses that controlled for total DNA content during transfection (data not shown). Our results demonstrate that inclusion of uncleaved MA-CA protein in HIV-1 particles results in a decrease in infection and that the residual level of infection is associated with altered sites of integration within the host genome.
TABLE 1.
Effects of uncleaved MA-CA on HIV-1 integration site preferences
| Library | No. of unique sites | No. (%) of sites within: |
Avg gene density within 1 Mb (±0.5 Mb) of integration sitesb | |||
|---|---|---|---|---|---|---|
| RefSeq genesa | 5 kb (±2.5 kb) of TSSa | 5 kb (±2.5 kb) of CpG islanda | 5 kb (±2.5 kb) of LADa | |||
| WT | 4,495 | 3,653 (81.3) | 222 (4.9) | 276 (6.1) | 774 (17.2) | 21.5 |
| 5% MA-CA plasmid | 1,094 | 846 (77.3)c | 51 (4.7)e | 56 (5.1)g | 251 (22.9)i | 18.6k |
| 20% MA-CA plasmid | 205 | 157 (76.6)d | 11 (5.4)f | 14 (6.8)h | 46 (22.4)j | 18.6l |
| RICm | 31,846 | 14,807 (46.5) | 1,710 (5.4) | 1,932 (6.1) | 13,916 (43.7) | 9.5 |
Statistical comparisons performed by Fisher’s exact test.
Statistical comparisons performed by Wilcoxon rank-sum test.
P value versus WT, 3.79 × 10−3.
P value versus WT, 1.87 × 10−1.
P value versus WT, 7.55 × 10−1.
P value versus WT, 8.27 × 10−1.
P value versus WT, 2.25 × 10−1.
P value versus WT, 9.22 × 10−1.
P value versus WT, 1.90 × 10−5.
P value versus WT, 5.49 × 10−2.
P Value versus WT, 3.58 × 10−12.
P value versus WT, 1.65 × 10−4.
RIC, random integration control.
Genetic determinants for MA-CA transdominance.
We sought to identify genetic determinants in Gag that are required for MA-CA transdominance. Based on the eccentric morphology of MA-CA-containing particles, Lee and coworkers suggested that the MA domain of MA-CA anchors the core to the viral membrane during assembly owing to its membrane-binding activity (39). The Gag polyprotein is myristoylated, which promotes membrane association and which is necessary for particle formation (67, 68). The membrane association of Gag also involves electrostatic interactions of the N-terminal basic residues in the MA region with acidic phospholipids in the cell membrane (69). Therefore, it is plausible that MA-CA-induced membrane tethering requires one or both of the membrane-binding elements in MA. To test this, we created and tested MA-CA plasmids lacking either or both elements. The protein changes included the N-terminal G2A substitution in MA (producing the MA-CA G2A construct), which prevents myristoylation of Gag, and two large internal deletions in MA that remove the N-terminal basic patch and that are compatible with particle assembly: deletion of codons 8 to 126 (Δ8-126 MA-CA) and deletion of codons 8 to 87 (Δ8-87 MA-CA) (70). We observed that HIV-1 particles containing uncleaved MA-CA proteins lacking these regions were as impaired for infection as those containing the full-length uncleaved MA-CA protein across the range of cotransfections (Fig. 9A). Therefore, the majority of the coding region of MA, including the N-terminal basic patch (KKQYKLKH), is not necessary for MA-CA antiviral activity.
FIG 9.
MA-CA antiviral potency requires membrane-binding elements in Gag. (A to C) Wild-type plasmid DNA was cotransfected with MA-CA plasmids encoding the indicated substitutions or deletions in MA. Infectivity is shown relative to that for the control virus (the 0% mutant construct). Shown are the mean values from three to six independent experiments. Error bars represent standard deviations. Significance was analyzed by an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) CA immunoblot of mutant particles for which the results are shown in panels A and C. (E) CA immunoblot of the mutant particles produced by transfection with plasmids encoding substitutions preventing cleavage at MA-CA or CA-NC junctions. 50/50, particles produced by cotransfection of equal quantities of the MA-CA and CA-NC mutant constructs. Lanes 4 to 8 contain dilutions of mutant particles produced by transfection of an HIV-1 plasmid encoding both MA-CA and CA-NC substitutions, resulting in the production of an uncleaved Gag protein extending from MA through NC.
To test whether myristoylation of Gag is required, we assayed the infectivity of HIV-1 particles generated by cotransfection of the wild-type and the myristoylation-defective MA-CA (MA-CA G2A) HIV-1 constructs. The resulting mixed particles were as impaired for infection as those containing the uncleaved MA-CA protein (Fig. 9B). Strong inhibition by the MA-CA G2A construct required the Y132I substitution, as the construct encoding only the G2A substitution was only mildly inhibitory, as previously reported (39). These results indicate that myristoylation is not necessary for MA-CA antiviral activity.
Finally, to test for the requirement of both membrane-binding elements in MA for MA-CA transdominance, we generated mixed particles with the wild-type, Δ8-126 MA-CA G2A, and Δ8-87 MA-CA G2A constructs. While both nonmyristoylated deletion constructs inhibited HIV-1 infectivity, the antiviral potency was 3- to 20-fold less than that of full-length MA-CA (Fig. 9C). The Δ8-87 MA-CA G2A protein was incorporated into particles at MA-CA/CA ratios similar to the ratio for full-length MA-CA (Fig. 9D). The Δ8-126 MA-CA G2A protein was also incorporated into particles. However, removal of the region from codons 8 to 126 from the uncleaved MA-CA protein resulted in a protein that we were unable to resolve from CA by SDS-PAGE. Collectively, these results indicate that at least one membrane-binding element of MA is necessary for the antiviral potency of uncleaved MA-CA.
Experiments involving cotransfections of two highly similar plasmids could be affected by host cell-mediated DNA recombination (71). In principle, recombination between the wild-type and the MA-CA G2A plasmid DNAs could result in segregation of the G2A and Y132I substitutions, potentially confounding the interpretation of the results shown in Fig. 9. To determine if recombination was a significant factor in our genetic studies, we cotransfected the MA-CA plasmid with a plasmid harboring substitutions (L363I/M367I/M377V) that inhibit cleavage between CA and NC (CA-NC) (72). In this assay, we cotransfected the plasmids at a 1:1 ratio to maximize the probability of recombination. If recombination occurred within the CA-coding region of the plasmids, it would result in a DNA molecule harboring all four mutations, resulting in particles containing a 48-kDa MA-NC protein. Immunoblotting analysis showed that particles resulting from the cotransfection contained only trace amounts of a band corresponding to uncleaved MA-NC. Similar quantities of this band were observed in particles produced by transfection of either the MA-CA or the CA-NC plasmid alone, suggesting that it did not result from recombination (Fig. 9E). These results suggest that DNA recombination in our cotransfection experiments was inefficient and therefore did not result in significant expression of uncleaved MA-CA protein that could be myristoylated.
DISCUSSION
In this study, we employed biochemical and cell-based assays to dissect the mechanism by which a substoichiometric amount of uncleaved MA-CA protein potently interferes with HIV-1 infectivity. Using a disulfide cross-linking approach and in vitro immunoprecipitation, we observed evidence for coassembly between CA and MA-CA in particles, and we observed the cosedimentation of the MA-CA protein with the genome and core proteins. We also showed that uncleaved MA-CA inhibits infection at nuclear entry and alters integration targeting. Finally, we showed that removal of both of the membrane-binding elements of MA reduced the antiviral potency of uncleaved MA-CA.
Postentry events in the HIV-1 life cycle include reverse transcription, uncoating, intracellular transport, entry into the nucleus, and integration into host chromatin. The role of the capsid during all of these stages has been the subject of numerous studies (for reviews, see references 5 and 73, to ,76). However, a well-defined model of the spatial and temporal aspects of uncoating has yet to emerge. Several genetic studies have coupled the intrinsic stability of the capsid to reverse transcription, nuclear entry, and infectivity (44, 45, 60, 77–81). The phenotypes that we observed in particles containing uncleaved MA-CA protein suggest that coassembly does not reduce the stability of the capsid: such particles exhibited normal levels of core-associated CA and nearly wild-type levels of late reverse transcription products, and, importantly, they retained the ability to abrogate restriction by TRIMCyp (44, 51, 54), which requires a stable capsid. However, our results do not preclude the possibility that uncleaved MA-CA hyperstabilizes the capsid lattice, potentially resulting in hyperstable cores that undergo delayed uncoating in target cells. This hypothesis is consistent with reports that CA substitutions that hyperstabilize the capsid generally result in infection defects manifested after reverse transcription (45, 60).
Both nuclear entry and integration targeting are affected by capsid binding to host cell factors (for reviews, see references 5 and 73 to 76), and incorporation of uncleaved MA-CA into the assembling viral capsid may affect these interactions, potentially accounting for our observation that these particles were impaired at nuclear entry and exhibited altered integration targeting. Our experiments showed that inclusion of MA-CA marginally decreased integration into genes and gene-dense areas of the host chromatin and increased integration near heterochromatic LAD regions. While 5% MA-CA viruses scored significantly differently from the WT virus across these metrics, we suspect that 20% MA-CA viral infections failed to attain statistical significance for genes and LAD regions due to the comparatively fewer number of integration sites recovered (Table 1). The MA-CA viral phenotype is reminiscent of what occurs with WT virus when host factor Nup153 (57, 82) or Nup358 (62) is depleted from the target cells. In both cases, a primary effect is observed at the step of preintegration complex nuclear import (83, 84), which is accompanied by significant integration retargeting away from gene-dense chromatin regions (57, 62, 82). This basic phenotype, moreover, is observed upon restriction of WT virus by the antiviral protein MxB (61). Although MxB is unlikely to be expressed at tangible levels under the conditions of viral infection used herein, recent reports have implicated components of the cellular nuclear import machinery in the mechanism of MxB antiviral activity (85, 86). We accordingly propose that analysis of the effects of MA-CA coassembly on Nup153 and Nup358 binding to CA in vitro or in cells in the presence or absence of MxB may help to inform the molecular mechanism of altered MA-CA integration site targeting observed here.
The eccentric particle morphology defect associated with MA-CA-containing virions is reminiscent of the morphology defect caused by exposure of HIV-1-producing cells to ALLINIs or by class II IN mutations (28–35). Despite this similarity, the resultant virus infection phenotypes are seemingly quite different. While the IN-perturbed viruses are defective for reverse transcription (28–35) due to the uncoupling of the vRNP from the capsid shell (87, 88), defective virus that harbored 20% MA-CA was largely proficient for reverse transcription (Fig. 7D). Possibly, the proposed tethering of MA-CA to the viral membrane (39) helps to stave off the rapid loss of the vRNP after virus entry, allowing reverse transcription to proceed largely unfettered. Indeed, our genetic studies revealed a dependence on membrane-binding elements in MA for the antiviral potency of uncleaved MA-CA. Coupled with our observation that MA-CA coassembles with CA and is present in the viral core, we suspect that the core structure remains attached to cellular membranes after virus entry, which could obscure efficient trafficking to the nucleus. We speculate that upon completion of reverse transcription, the preintegration complex (PIC) dissociates from the cellular membranes, whereby the MA-CA component of the PIC may then interfere with CA-Nup interactions, obstructing nuclear entry and integration targeting. We accordingly suggest that both membrane tethering of the core and interference with capsid-Nup interactions contribute to the unique antiviral activity of core-associated MA-CA. Testing of the membrane tethering hypothesis will be an interesting future direction that will require the development of appropriate cell fractionation and/or live-cell imaging techniques.
Our observation that pseudotyping by VSV-G reduces the antiviral potency of uncleaved MA-CA supports the notion for the involvement of the target cell membrane in the antiviral mechanism. Pseudotyping by VSV-G targets HIV-1 entry to an endocytic route that requires exposure to the low endosomal pH for membrane fusion to be activated (55). Endocytic entry may reduce the inhibition by promoting endosomal transport of the virus within the cell prior to fusion. Additionally, exposure to the low pH of the endosome could facilitate the membrane detachment and/or capsid disassembly events required for infection. We suspect that a persistent membrane association may also provide a possible explanation for the reduced density of peak CA observed in cores isolated from particles containing uncleaved MA-CA protein (Fig. 5E). A strong association of uncleaved MA-CA protein with the viral membrane might result in the retention of lipids, despite the exposure to detergent during isolation of the cores, potentially reducing the density of the isolated cores (47). Another possible explanation for the altered density of MA-CA-containing cores is the apparent dissociation of the vRNP from the capsid shell in eccentric viral particles (Fig. 1C). HIV-1 isolates made in the presence of ALLINIs as well as class II IN mutant viruses, both groups of which harbor similar morphological defects, also yield similar changes in core density (28, 88). Cryo-ET analysis of MA-CA-containing virions may help to delineate aspects of the eccentric particle phenotype that differ from the phenotype induced by class II IN mutations or ALLINI exposure, both of which instill dramatic reverse transcription defects.
Immature HIV-1 particles reportedly contain 2,400 Gag molecules (89), while the mature capsid is comprised of approximately 1,500 CA subunits (7). Thus, proteolytic maturation results in an excess of CA molecules that are not incorporated into the mature capsid. Given the surplus of Gag subunits in particles, the approximately 480 subunits of uncleaved MA-CA present in a 20% mixed particle represent a pool of subunits that presumably are unnecessary for a mature capsid to form. Therefore, it is conceivable that the small amounts of uncleaved MA-CA protein present in mixed particles are not incorporated into the mature capsid structure, affecting the core in trans through random incorporation, possibly by interfering with normal CA functions. Nonetheless, our cross-linking analysis, in vitro binding reactions, and biochemical characterization of isolated cores provide evidence that uncleaved MA-CA coassembles with CA. One possible consequence of MA-CA coassembly with CA into the capsid is disruption of the intermolecular interfaces that construct the mature CA lattice, possibly explaining the morphological assembly defect observed by us and others (39). In a previous study, we observed that the HIV-1 CA-binding small-molecule Boehringer Ingelheim compound 1 induces aberrant cross-links at the CTD-CTD 3-fold axis in particles capable of disulfide cross-linking (90). However, we did not observe a similar effect of MA-CA capable of the same cross-linking. Additionally, while canonical recombinant 14C/45C CA tubes were not observed when coassembled with MA-CA, hexamer formation was not prohibited. Our results suggest that while MA-CA induces morphological assembly defects, the capsids in particles containing uncleaved MA-CA protein are hexameric lattices. This interpretation is also supported by our observation that the particles abrogate restriction by TRIMCyp (51, 54). Nonetheless, a hexameric lattice is not sufficient for a closed capsid structure, which requires inclusion of 12 CA pentamers, which we were unable to test using the engineered disulfide cross-linking approach.
Recently, the host small-molecule inositol hexakisphosphate (IP6) has been shown to bind both immature Gag and mature CA hexamers during the assembly of each lattice (91–94). Because cleavage at the MA-CA junction is required for β-hairpin formation, incorporation of MA-CA into the CA lattice may inhibit IP6 binding to the hexamer and undermine its potential effects, including modulation of capsid stability and binding of host proteins.
In summary, we have shown that small amounts of uncleaved MA-CA protein can coassemble with CA during HIV-1 maturation and that incorporation of uncleaved MA-CA inhibits HIV-1 nuclear entry and affects integration site targeting. Particles containing uncleaved MA-CA exhibit an aberrant morphology but have stable capsids and are competent for reverse transcription in target cells. We propose that the inclusion of MA-CA in the mature capsid lattice alters the capsid-host factor interactions necessary for HIV-1 nuclear import and integration targeting and that impaired nuclear entry is also exacerbated by the tethering of MA-CA to cellular membranes. The accumulation of uncleaved MA-CA may account, at least in part, for the marked reduction in HIV-1 infectivity observed under conditions of partial inhibition of the viral protease (23).
MATERIALS AND METHODS
Plasmids.
All viruses tested in this study were generated from pNL4-3 or R9 proviral clones, which are isogenic in the Gag-coding region. The pNL4-3 MA-CA plasmid harbors the single Y132I substitution in Gag (47). The following substitutions were generated by PCR overlap in pNL4-3 and pNL4-3 MA-CA constructs: in MA, G2A; in CA, A14C and E45C. The following deletions in MA were generated in wild-type, MA-CA, and MA-CA G2A pNL4-3 plasmids by overlap PCR: removal of codons 8 to 126 and codons 8 to 87. For constructs in which codons 8 to 126 and 8 to 87 were deleted, a Ser/Arg (TCTCGT) motif was inserted concomitantly with the removal in a single phase of overlap PCR. Table S1 in the supplemental material lists the oligonucleotides used for introducing the mutations. The methods used for the construction of the pNL4-3 CA-NC plasmid (72) and the pNL4-3 MA-NC plasmid (95) were described previously. pNL4-3ΔE encodes a frameshift at the NdeI restriction site in env, resulting in Env-deficient particles. Plasmid pNLX.Luc.R-U3-tag, which was derived from pNLX.Luc.R-.ΔAvrII using overlap PCR (with the primers listed in Table S1), is also env deficient and additionally carries the gene for firefly luciferase (Luc) in the viral nef position (57). The resulting PCR product, digested with XhoI and NgoMIV, was ligated with XhoI/NgoMIV-digested pNLX.Luc.R-.ΔAvrII DNA. The accuracy of mutagenesis was confirmed by DNA sequencing of all PCR-amplified regions.
Env-deficient viruses were pseudotyped with the vesicular stomatitis virus G protein (VSV-G) by cotransfection with pHCMV-G (96). Reporter-based infection assays employed HIV-GFP, a pNL4-3-based construct that harbors an env frameshift mutation and encodes green fluorescent protein (GFP) in place of nef (97), or HIV-Luc, generated from pNLX.Luc.R-.ΔAvrII. The BlaM-Vpr reporter viruses used in virus-cell fusion assays were generated by cotransfection with the pMM310 plasmid, as previously described (95).
The recombinant 14C/45C CA protein was generated by introduction of Cys codons into pET21a HIV-1 CA at positions 14 and 45 (42). The recombinant MA-CA protein was generated by introducing the MA-CA coding region of pNL4-3 into the pET21a vector via the restriction enzymes NdeI and XhoI.
Cells and viruses.
TZM-bl cells were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (catalog number 8129), from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. (56, 98–101). HeLa-P4 cells were the generous gift of F. Clavel (66). Owl monkey kidney (OMK) and HEK293T human embryonic kidney (HEK) cells were purchased from the American Type Culture Collection. HeLa-CD4+ cells were generated by transducing HeLa cells with a pBABE-puro (102) vector encoding CD4. Single-cell clones were grown, and cells were selected for growth with puromycin (1 μg/ml). All cells were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS), penicillin (50 IU/ml), and streptomycin (50 μg/ml). The virus stocks used for infectivity, cell fusion, abrogation of restriction, and protein cross-linking analyses were produced by calcium phosphate transfection of HEK293T cells (103). Virus stocks for thin-section EM analysis and the HIV-Luc virions used to test the infectivity of the U3-tagged virus for integration site sequencing were produced via PolyJet (SignaGen Laboratories) transfection (58). The virus stocks used for the isolation of cores and integration targeting analysis were generated by polyethylenimine (PEI) transfection owing to the need for higher particle yields. Briefly, 10 μg of plasmid DNA was added to 900 μl Opti-MEM (Gibco) and 40 μg PEI, and the mixture was incubated for 15 min at room temperature and subsequently added to 3 million HEK293T cells. The cells were incubated at 37°C in 5% CO2 for 6 h and subsequently washed in phosphate-buffered saline (PBS), and 6 ml medium was replaced. Cell supernatants were harvested at 48 h posttransfection. The viruses used for quantification of the reverse transcription products and 2 long terminal repeat (2-LTR) circles by qPCR were generated by transfection using the TransIT-293 transfection reagent (Mirus), according to the manufacturer’s protocol, to minimize contamination of the virus stocks by transfected plasmid DNA. For the cotransfection experiments, the total amount of DNA transfected was kept constant between experiments, using 20 μg total DNA per dish for calcium phosphate transfections, 10 μg total DNA for PEI transfections, and 15 μg total DNA for TransIT-293 transfections. VSV-G-pseudotyped HIV-1 particles were generated by cotransfection of 20 μg of proviral DNA with 5 μg of pHCMV-G DNA. Culture supernatants were filtered through 0.45-μm-pore-size syringe filters, and aliquots were frozen and stored at −80°C only once per use. The CA content of the virus stocks was quantified by a p24-specific enzyme-linked immunosorbent assay (ELISA) (104), where viral samples were initially diluted in 2× Laemmli buffer (347 mM Tris-HCl [pH 6.8], 22% glycerol, 0.0167% bromophenol blue, 4.4% sodium dodecyl sulfate [SDS]) containing 10% 2-mercaptoethanol (BME) and heated to 95°C for 5 min prior to serial dilution.
Virion morphology analysis.
HIV-1 particles produced by transfection were pelleted at 26,000 rpm (115,000 × g) at 4°C for 2.5 h with a Beckman SW32 Ti rotor. The viral pellets were collected in 1 ml of PBS and subsequently pelleted at 45,000 rpm (125,000 × g) at 4°C for 30 min in a Beckman TLA 55 rotor. Viral pellets were fixed overnight at 4°C in electron microscopy (EM)-grade (Electron Microscopy Sciences) fixative agents (1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% picric acid, 0.1 M sodium cacodylate buffer [pH 7.4]). Ultrathin sections (∼60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate, and observed using a JEOL 1200EX microscope with an AMT 2k charge-coupled-device camera. Images were captured at a ×30,000 magnification and were visually inspected to classify viral particles as mature, immature, eccentric, or empty. Virus particle classification was conducted using samples to which the investigators were blind to the identities, and over 100 particles were counted per virus preparation. Linear regression analysis of the eccentric particles was performed using GraphPad Prism software.
Virus-cell fusion assay.
Viral fusion was assayed as described before (95), with modification. Briefly, we inoculated 10,000 HeLa TZM-bl cells, which had been seeded the day before, in a black-walled 96-well plate with dilutions of BlaM-Vpr reporter viruses in DMEM that was free of FBS, penicillin, and streptomycin but that contained HEPES (10 mM) and DEAE-dextran (20 μg/ml). Diluted viruses (100 μl) were added to the cells, and the cells and viruses were cultured for 2 h at 37°C to allow for virus fusion. CCF4-AM (Invitrogen) was then added to a final concentration of 0.94 μM, and the mixture was incubated at room temperature for 16 h in the dark. Supernatants were removed and replaced with 100 μl PBS, and the fluorescence was quantified at 450 nm and 520 nm with a BMG Fluostar microplate fluorometer with excitation at 410 nm. The extent of fusion was determined as the resulting ratio of blue-to-green fluorescence in each culture. These ratios were calculated following subtraction of the averaged background values for uninoculated (blue values) and cell-free, PBS-containing quadruplicate wells (green background). Duplicate determinations were performed for each virus dilution.
Assay of HIV infectivity.
Viral infectivity was determined by titrating virus stocks on TZM-bl cells and by assaying the expression of luciferase reporter activity in cell lysates or by using p24-matched levels of luciferase-containing constructs to infect HEK293T cells (58). Cells (10,000 per well) were seeded in black-walled 96-well plates and inoculated with 10-fold serial dilutions of HIV-1NL4-3 in culture medium containing 20 μg/ml DEAE-dextran in a 100-μl total volume. At 48 h after inoculation, the cells were washed once in PBS and lysed in 30 μl Tris-buffered saline (50 mM Tris-HCl [pH 7.8], 130 mM NaCl, 10 mM KCl, 5 mM MgCl2) containing 0.5% Triton X-100. Luminescence was determined using an Lmax luminometer (Molecular Devices) for 5 s after injection of 200 μl solution 1 (75 mM Tris-HCl [pH 8], 8.3 mM magnesium acetate, 4 mM ATP) and 80 μl solution 2 (1 mM d-luciferin [GoldBio]). Infectivity values were determined as the ratio of arbitrary light units (ALU) per nanogram of p24 at doses of virus corresponding to the linear range of the luciferase signals from each assay. The infectivity of particles containing uncleaved MA-CA was calculated as a percentage of the corresponding wild-type values. For abrogation of restriction experiments, single-cycle assays of viral infectivity were performed with 10,000 OMK cells plated 1 day prior to infection in 96-well plates with 100-μl volumes of HIV-1ΔE(VSV), coinoculated with HIV-GFP(VSV) (2 ng p24). At 16 h after inoculation, the cells were washed in PBS and the culture medium was replaced. At 48 h postinfection, the cells were detached with trypsin and fixed overnight in PBS containing 4% fresh paraformaldehyde. GFP expression in OMK cells was analyzed by flow cytometry on an Accuri C6 flow cytometer, analyzing a minimum of 5,000 cells per sample.
Isolation of HIV-1 cores.
Viral cores were isolated from concentrated HIV-1 particles as previously described (46). Supernatants from transfected HEK293T cells were concentrated by centrifugation in a Beckman SW32-Ti rotor at 32,000 rpm (175,000 × g at maximum radius) at 4°C for 3 h. The pelleted virions were resuspended in 0.4 ml of 1× STE buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA) and incubated on ice for 3 h. The concentrated virions were then centrifuged at 32,000 rpm (187,000 × g) for 16 h at 4°C through an 11-ml 30% to 70% linear sucrose gradient overlaid with 1% Triton X-100. Fractions (1 ml) were collected from the top of the gradient. The CA content in each fraction was quantified by a p24 ELISA. The HIV-1 RNA in each fraction was quantified by RT-qPCR. Prior to concentration of the particles and core isolation, transfected cell supernatants were treated with 20 μg/ml DNase I and 10 mM MgCl2 for 1 h at 37°C. RNA was isolated from each fraction with the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized from the extracted RNA by RT-PCR using a cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. cDNA from each fraction was analyzed by quantitative PCR using an MX-3000P thermocycler (Stratagene) utilizing SYBR green chemistry (ABI). First-strand-transfer reverse transcripts were amplified using forward (5′-AGCAGCTGCTTTTTGCCTGTACT-3′) and reverse (5′-ACACAACAGACGGGCACACAC-3′) primers, and copy numbers were interpolated from a standard curve produced with dilutions of wild-type R9 plasmid DNA. The HIV-1 proteins in each gradient fraction were analyzed by immunoblotting after concentrating the protein samples by precipitation with trichloroacetic acid (TCA). Briefly, 500 μl of the gradient fractions was diluted in 500 μl PBS. Two hundred fifty microliters of 30% TCA was added, and samples were incubated on ice for 30 min. Precipitated proteins were pelleted at 13,000 rpm for 5 min. The pellets were washed twice in a 1:1 ethanol-ether mix. The pellets were then resuspended in Laemmli buffer.
Immunoblotting analysis.
Proteins were separated on 4% to 20% SurePAGE bis-Tris polyacrylamide gradient gels (GenScript) and electrophoretically transferred to nitrocellulose membranes (PerkinElmer). The blots were subsequently blocked in blocking buffer (LI-COR Biosciences) diluted 50% with PBS. The membranes were washed in PBS containing 0.2% Tween 20 thrice for 5 min each time between antibody incubations. For immunoblotting analysis of pelleted viral particles, samples were normalized by the p24 levels (determined by ELISA), with the proteins being dissolved in 2× Laemmli buffer containing 10% BME and incubated for 5 min at 95°C (reducing conditions) or dissolved in Laemmli buffer without BME and not heated (nonreducing conditions). Samples from TCA-precipitated gradient fractions were loaded onto gels with equal volumes between samples. The membranes were probed with the following antibodies: HIV-1 CA (4 μg/ml; mouse monoclonal antibody; catalog number 183-H12-5C; NIH AIDS Research and Reference Program [105]), HIV-1 NC (1:1,000 dilution; goat serum polyclonal antibody; received from Robert Gorelick), HIV-1 MA (1:500 dilution; rabbit serum polyclonal antibody; catalog number 4811; NIH AIDS Research and Reference Program [106]), and HIV-1 RT (1:1,000 dilution; rabbit serum polyclonal antibody; catalog number 6195; NIH AIDS Research and Reference Program). Rabbit antiserum against HIV-1 IN was raised in animals inoculated with 2 μg purified recombinant protein (107), which was subsequently affinity purified over an IN-containing column (Thermo Fisher). The resulting antibody was used at a 1:5,000 dilution. BlaM-Vpr was detected with beta-lactamase antibody (2.8 μg/ml; mouse monoclonal antibody; catalog number 15720; QED Bioscience Inc.). The immunoblots were probed with the appropriate infrared (IR) dye-conjugated secondary antibodies at 100 ng/ml (LI-COR Biosciences), and bound antibodies were detected by scanning with a LI-COR Biosciences Odyssey imaging system. Protein band quantification was determined via pixel intensity analysis using LI-COR Biosciences Image Studio (version 3.1) software, employing background subtraction by the top-and-bottom, median background method.
Quantification of reverse-transcribed products and 2-LTR circles in infected cells.
Duplicate cultures of 100,000 HeLa-CD4 cells were inoculated with 150 ng p24 of HIV-1 particles in 12-well plates. Cell monolayers were rinsed in PBS and detached with trypsin at 8 h after virus inoculation (for second-strand transfer analysis) or 24 h postinoculation (for 2-LTR-circle analysis). Cell pellets were treated with qPCR lysis buffer (10 mM Tris-HCl [pH 8], 0.2 mM CaCl2, 1 mM EDTA, 0.001% Triton X-100, 0.001% SDS, 1 mg/ml proteinase K) for 1 h at 57°C. The proteinase K was then inactivated by heating the samples at 95°C for 15 min. As a control to detect contaminating plasmid DNA, the RT inhibitor efavirenz (1 μM) was added to parallel infections in all experiments analyzing second-strand transfer. Either 25 μM IN inhibitor 118-D-24 (NIH AIDS Research and Reference Program [108, 109]) or 1 μM raltegravir (a generous gift from Chandravanu Dash) was added to all samples in experiments analyzing 2-LTR circles. Prior to all infections for qPCR analysis, virus stocks were treated with 20 μg/ml DNase I, 10 mM MgCl2 at 37°C for 1 h to reduce contaminating plasmid DNA. HIV-1 DNA was detected in each sample with SYBR green chemistry on a Stratagene MX3000p instrument. Second-strand transfer DNA was amplified using forward primer 5′-TGTGTGCCCGTCTGTTGTGT-3′ and reverse primer 5′-GAGTCCTGCGTCGAGAGATC-3′. 2-LTR-circle DNA was amplified using forward primer 5′-AACTAGGGAACCCACTGCTTAAG-3′ and reverse primer 5′-TCCACAGATCAAGGATATCTTGTC-3′. Second-strand-transfer copy numbers of cDNA were interpolated from a standard curve using HIV-1 plasmid DNA as a standard. 2-LTR-circle copy numbers of cDNA were interpolated from a standard curve using p2LTR plasmid DNA (110) as a standard.
Recombinant protein purification and immunoprecipitation.
The HIV-1 proteins MA-CA and CA were expressed from the pET21a-MA-CA construct and the pET21a-CA construct, which harbors A14C/E45C substitutions in CA. The plasmids were transformed into competent Escherichia coli BL21 cells, which were grown in 2 liters of LB to an optical density at 600 nm of 0.6; expression was induced by the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside; Gold Biotechnology); and the cultures were subsequently incubated for 8 h at 25°C. Cells were lysed by sonication (Sonic Dismembrator ultrasonic processor; Fisher Scientific), and the lysates were cleared by centrifugation for 1 h at 40,000 × g at 4°C. The CA protein was purified by anion-exchange chromatography as described previously (42). The MA-CA protein was purified as described previously (14), with modification. The MA-CA protein was precipitated from the bacterial lysate by addition of ammonium sulfate to 25% to 34% of saturation, and the precipitate was pelleted and dissolved in 30 ml buffer A (25 mM Tris-HCl [pH 8.0], 50 mM NaCl, 5 mM BME, protease inhibitor cocktail tablet [Roche]), dialyzed against 1 liter of buffer A overnight at 4°C, clarified by centrifugation for 30 min at 27,000 × g, and then subjected to anion-exchange chromatography using a 5 ml UNOsphere Q cartridge (Bio Rad). The MA-CA protein was present in the flowthrough and subsequently spontaneously precipitated. The precipitate was pelleted and dissolved in 5 ml of STE buffer containing 5 mM BME and 0.2% dodecyl phosphocholine (DPC). DPC was removed by two rounds of dialysis against 1 liter of 50 mM Tris-HCl, pH 7.4, 1 M NaCl at 4°C for 16 h, and the sample was subsequently concentrated to 0.8 ml with an Amicon Utracel-10K filter unit (Millipore). The final yield of MA-CA protein was approximately 3 mg, and the purity was greater than 95%, as determined by SDS-polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie blue.
Recombinant CA and MA-CA proteins were assembled in 50 mM Tris-HCl (pH 8.0), 1 M NaCl at 37°C for 1 h at 0.8 mg/ml each, and the assembled proteins were pelleted by centrifugation at 5,000 × g for 5 min at 4°C. The supernatant was removed, and the pelleted assembled protein was resuspended in 25 mM Tris-HCl (pH 8.0), 0.5 M NaCl. Immunoprecipitation was performed with Pierce protein A/G magnetic beads (Thermo Scientific) per the manufacturer’s instructions. The beads (200 μg) were coated with 5 μl anti-MA antibody (rabbit polyclonal antibody; catalog number 4811; NIH AIDS Research and Reference Program) for 1 h at room temperature in wash buffer (25 mM Tris-HCl [pH 8.0], 0.5 M NaCl, 0.05% Tween 20). Fifteen microliters of each resuspended assembly or coassembly reaction mixture was added to the coated beads, and the mixture was incubated for 16 h at 4°C in 25 mM Tris-HCl (pH 8.0), 0.5 M NaCl. The beads were washed thrice in wash buffer. The beads were resuspended in 30 μl of 2× Laemmli buffer containing 10% BME and incubated for 10 min at 95°C. The beads were removed from the sample by use of a magnet prior to SDS-PAGE and immunoblotting.
Visual inspection of recombinant CA and MA-CA assembly reactions.
Recombinant CA and MA-CA proteins were assembled as described above at 0.8 mg/ml each and pelleted, the supernatants were removed, and the pelleted reaction mixtures were resuspended in 25 mM Tris-HCl (pH 8.0), 0.5 M NaCl. To check tube assembly, the reactions were visualized by negative-stain EM after staining with 0.7% uranyl formate, and images were collected with an FEI Morgagni electron microscope at 100 kV at a magnification of ×4,400 or ×2,200. Images were recorded on an AMT 100 × 100 charge-coupled-device camera.
Integration site analysis.
In preliminary experiments, genomic DNA was extracted from HEK293T cells infected with VSV-G-pseudotyped HIV-1NLX.Luc.R- or HIV-1NLX.Luc.R-U3-tag using a DNeasy blood and tissue kit (Qiagen). To sequence U5-cell DNA junctions, genomic DNA was digested with MseI and BglII (65). For U3-cell junctions, DNA isolated from cells infected with HIV-1NLX.Luc.R-U3-tag was digested with AvrII, NheI-HF, SpeI-HF, and BamHI-HF. Following digestion, fragmented DNA was ligated to adapters and subjected to consecutive rounds of ligation-mediated PCR (LM-PCR), essentially as previously described (65) (see Table S1 for the primers utilized) using 16 independent PCRs for each primer pair during both rounds of PCR.
Plasmid pNLX.Luc.R-U3-tag was cotransfected with either the pNL4-3 or the pNL4-3 MA-CA plasmid and pHCMV-G to produce pseudotyped HIV-1 particles, which were then used (12.5 ng of p24) to inoculate 0.5 million HeLa-P4 cells. The cells were subsequently cultured for 4 days, and cellular DNA was extracted (Qiagen DNeasy blood and tissue kit). The DNA was processed for LM-PCR and Illumina sequencing essentially as described above and as described in references 59 and 65. Read pairs from 150-bp paired-end sequencing were selected for U3 LTR sequences in the first read (read 1) and linker DNA in the second read (read 2). After trimming both the LTR and linker sequences, the reads were aligned to human genome build hg19 by use of the Burrows-Wheeler aligner (BWA) BWA-MEM with the paired-end option (111). The MA-CA virus experiments compiled the results from two independent infections, and the alignment outputs from both experiments were combined to generate single-alignment files to map integration sites. For preliminary work with the U3-tagged virus, only read 1 information was aligned using the Hisat aligner (112). BWA-aligned reads were filtered to remove unmapped reads using the –F 4 option in SAMtools (113), while secondary alignment and reads with low MAPQ scores were removed using the –F 256 –q 1 options. The paired-end analysis filtered for reads that were properly distanced and directed toward each other on the same chromosome; reads where the distance between the integration site and the linker ligation site was >900 bp were omitted from analysis. Unique reads across pipelines were selected and converted into the BED format. To account for the 5-bp duplication of genomic DNA associated with HIV-1 integration, the left interval of the BED format was determined by adding 2 to the integration site if the site was on the genomic positive strand and by subtracting 2 from the site if it was on the negative strand. Similarly, the right interval of the BED format was obtained by adding 3 to the integration site if the site was on the positive strand and by subtracting 3 if the site was on the negative strand. The distribution of integration sites with respect to various genomic features of the human genome was analyzed using the BEDtools suite of tools as described previously (65). The computation random integration control (RIC) was similarly mapped using DNA fragments generated following the digestion of hg19 with the restriction endonucleases AvrII, NheI, Spe, and BamHI in silico. Statistical analyses were performed as described previously (65).
Data availability.
FASTQ files for integration site data sets have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the following accession numbers: PRJNA552541 (for the U3-tagged vector in HEK293T cells) and PRJNA552059 (for MA-CA viral samples in HeLa-P4 cells).
Supplementary Material
ACKNOWLEDGMENTS
We thank Jiong Shi for assistance with sample preparation and Jing Zhou for preparation of the recombinant MA-CA protein. We thank Eric Poeschla (the University of Colorado School of Medicine) for advice on the design of the U3-tagged construct for integration site sequencing. Raltegravir was a kind gift from Chandravanu Dash, Meharry Medical College. We thank Robert Gorelick, NCI, for the anti-NC antibody. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: anti-HIV-1 p24 hybridoma (clone 183-H12-5C; catalog number 1513) was from Bruce Chesebro, anti-HIV-1 p17 polyclonal antibody (catalog number 4811) was from Paul Spearman and Lingmei Ding, anti-HIV-1 RT polyclonal antibody (catalog number 6195) was from Stuart LeGrice, integrase inhibitor (catalog number 118-D-24) was from NIAID, Division of AIDS (catalog number 9957), and TZM-bl cells (catalog number 8129) were from John C. Kappes and Xiaoyun Wu.
This work was supported by NIH grant P50 AI150481 to the Pittsburgh Center for HIV-Protein Interactions.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JVI.01118-19.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
FASTQ files for integration site data sets have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the following accession numbers: PRJNA552541 (for the U3-tagged vector in HEK293T cells) and PRJNA552059 (for MA-CA viral samples in HeLa-P4 cells).