Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.     

Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli

Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coli were examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.     

Increased assembly of clathrin occurs in response to mitogenic activation of murine lymphocytes

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.     

Reversible binding of recombinant human immunodeficiency virus type 1 gag protein to nucleic acids in virus-like particle assembly in vitro

Recombinant human immunodeficiency virus type 1 (HIV-1) Gag protein can assemble into virus-like particles (VLPs) in suitable buffer conditions with nucleic acid. We have explored the role of nucleic acid in this assembly process. HIV-1 nucleocapsid protein, a domain of Gag, can bind to oligodeoxynucleotides with the sequence d(TG)(n) with more salt resistance than to d(A)(n) oligonucleotides. We found that assembly of VLPs on d(TG)(n) oligonucleotides was more salt resistant than assembly on d(A)(n); thus, the oligonucleotides do not simply neutralize basic residues in Gag but provide a binding surface upon which Gag molecules assemble into VLPs. We also found that Gag molecules could be "trapped" on internal d(TG)(n) sequences within 40-base oligonucleotides, rendering them unable to take part in assembly. Thus, assembly on oligonucleotides requires that Gag proteins bind near the ends of the nucleic acid, and binding of Gag to internal d(TG)(n) sequences is apparently cooperative. Finally, we showed that nucleic acids in VLPs can exchange with nucleic acids in solution; there is a hierarchy of preferences in these exchange reactions. The results are consistent with an equilibrium model of in vitro assembly and may help to explain how Gag molecules in vivo select genomic RNA despite the presence in the cell of a vast excess of cellular mRNA molecules.     

Nucleic acid-independent retrovirus assembly can be driven by dimerization

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.     

Characterization of Rous sarcoma virus Gag particles assembled in vitro

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.     

A cell-line-specific defect in the intracellular transport and release of assembled retroviral capsids

Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled form (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the development of a recombinant baculovirus/insect cell system for the expression of both C-type and D-type Gag polyproteins, we discovered an insect cell line (High Five) with two distinct defects that resulted in the reduced release of virus-like particles. The first of these was a pronounced defect in the transport of D-type but not C-type Gag polyproteins to the plasma membrane. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) Gag precursors accumulate assembled immature capsids in large cytoplasmic aggregates similar to a transport-defective mutant (MA-A18V). In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogenesis mutant (MA-R55W) and those of human immunodeficiency virus were transported to the plasma membrane for assembly and budding of virions. When pulse-labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequestered in a higher-molecular-mass complex. Compared to Sf9 insect cells, the High Five cells also demonstrate a defect in the release of C-type virus particles. These findings support the hypothesis that host cell factors are important in the process of Gag transport and in the release of enveloped viral particles.     

The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles

The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.     

Assembly Properties of Human Immunodeficiency Virus Type 1 Gag-Leucine Zipper Chimeras: Implications for Retrovirus Assembly

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant “Gag-Zipper” proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the “dimerizing” Gag-Zipper protein misassembles into very small particles, while the “trimerizing” protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the “dimerizing” Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.Expression of a single retroviral protein, Gag, in mammalian cells is sufficient for assembly of virus-like particles (VLPs). RNA seems to play an essential role, however, in both the assembly and structure of VLPs. Thus, retrovirus particles always contain RNA; in the absence of genomic RNA, cellular mRNAs replace it in the virus particle (46). RNase treatment of immature murine leukemia virus disrupts the particles (37). Finally, nucleic acid is required for assembly in defined in vitro assembly systems (8, 9).The contribution of nucleic acid to the assembly and structure of retrovirus particles is not yet understood. As one approach to further understanding the role that nucleic acid binding plays in the assembly process, Zhang et al. (59) replaced the principal nucleic acid-binding domain of the HIV-1 Gag protein, nucleocapsid (NC), with a leucine zipper domain. This chimeric protein was able to assemble efficiently in mammalian cells as evidenced through immunoblotting of released VLPs. This observation was extended by Johnson et al. (28), who used Gag-leucine zipper (dimerizing) chimeras of Rous sarcoma virus and studied the morphologies of the resulting particles. The particles assembled from the chimeric proteins were similar, although not identical, to those formed by wild-type (WT) Gag. The fact that NC could be functionally replaced (with respect to particle assembly) with the dimerizing leucine zipper motif led these investigators to propose that the function of nucleic acid in assembly is to promote dimerization. Additional support for this hypothesis comes from the fact that the minimum length of nucleic acid needed to promote assembly is roughly enough to accommodate two molecules of Gag (30, 31).Further studies in which the NC domain of HIV-1 Gag has been replaced by leucine zipper motifs have been presented by Accola et al. (1). Interestingly, they found that a Gag-Zipper (Gag-Z) chimera containing a trimeric zipper motif also assembles efficiently. However, these VLPs, as well as those formed by a chimera containing a dimeric zipper motif, were not characterized morphologically.In the present work, we have extended the analysis of the assembly properties of these HIV-1 Gag-Z chimeras. This study includes the first analysis of recombinant Gag-Z proteins in vitro, as well as detailed characterization of the VLPs formed in mammalian cells. The in vitro assembly results suggest that Gag oligomerization alone is not sufficient to induce particle formation. We raise the possibility here that normal HIV-1 assembly requires neutralization of positive charges in matrix (MA) in addition to nucleic acid-induced oligomerization at the C terminus of the protein.     

Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells.

The human immunodeficiency virus type 1 (HIV-1) Pr55gag precursors were previously shown to assemble and bud efficiently as noninfectious virus-like particles (VLPs) when expressed in baculovirus-infected insect cells. In this study, we examined the abilities of foreign antigens to be incorporated on the outer surface of HIV-1 Gag particles. We have used a dual recombinant baculovirus, expressing the HIV-1 Gag gene and gD gene under the control of the P10 and polyhedrin promoters, respectively, to obtain hybrid VLPs. Transmission electron microscopy of insect cells infected with the dual recombinant revealed very large aggregates of particles budding from the cell membrane. The release of VLPs into the culture medium was clearly different for a recombinant baculovirus producing solely HIV-1 Gag, for which particles were uniformly distributed all around the cell surface. Biochemical analysis of hybrid particles indicated that glycoprotein gD was packaged into HIV-1 Gag VLPs. Moreover, the carboxy-terminal p6 region of Gag polyprotein and the glycoprotein gD intracytoplasmic domain were not required for gD incorporation. The experiments described here clearly demonstrate that glycoprotein gD can be packaged with HIV-1 Gag particles and released from insect cells.     

A unique monoclonal antibody that recognizes mature p17 of HIV-1 but not its precursor.

The entire and partial gag regions of human immunodeficiency virus type 1 (HIV-1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17. We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety. Further analysis revealed that V17 recognizes the C-terminal 12-amino-acid region of p17 having free C-terminus. This monoclonal antibody may be useful for monitoring the maturation of virus particles.     

Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.     

Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro

As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag (deltaMBDdeltaPR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of deltaMBDdeltaPR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of deltaMBDdeltaPR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.     

Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates mainly with viral particles containing unprocessed gag

Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.     

Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR—a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.     

Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.