Separate Assembly and Transport Domains within the Gag Precursor of Mason-Pfizer Monkey Virus

Mason-Pfizer monkey virus (M-PMV), the prototypical type D retrovirus, assembles immature capsids within the cytoplasm of the cell prior to plasma membrane interaction. Several mutants of M-PMV Gag have been described which display altered transport, assembly, or both. In this report, we describe the use of an in vitro synthesis and assembly system to distinguish between defects in intracellular transport and the process of assembly itself for two previously described gag gene mutants. Matrix domain mutant R55W converts the type D morphogenesis of M-PMV particles into type C and has been hypothesized to alter the transport of Gag, redirecting it to the plasma membrane where assembly subsequently occurs. We show here that R55W can assemble in both the in vitro translation-assembly system and within inclusion bodies in bacteria and thus has retained the capacity to assemble in the cytoplasm. This supports the concept that R55 is located within a domain responsible for the transport of Gag to an intracellular site for assembly. In contrast, deletions within the p12 domain of M-PMV Gag had previously been shown to affect the efficiency of particle formation such that under low-level expression conditions, Gag would fail to assemble. We demonstrate here that the efficiency of assembly in the in vitro system mirrors that seen in cells under expression conditions similar to that of an infection. These results argue that the p12 domain of this D-type retrovirus plays a critical role in the membrane-independent assembly of immature capsids.     

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.     

Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.     

1H, 13C, and 15N resonance assignment of the N-terminal domain of Mason-Pfizer monkey virus capsid protein, CA 1-140

Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignment of the M-PMV NTD CA.     

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.     

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.     

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.     

Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.     

Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.     

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.     

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.     

Human immunodeficiency virus type 1 capsid formation in reticulocyte lysates.

The Gag polyprotein of human immunodeficiency virus (HIV) (Pr55Gag) contains sufficient information to direct particle assembly events when expressed within tissue culture cells. HIV Gag proteins normally form particles at a plasma membrane assembly site, in a manner analogous to that of the type C avian and mammalian leukemia/sarcoma viruses. It has not previously been demonstrated that immature HIV capsids can form without budding through an intact cellular membrane. In this study, a rabbit reticulocyte lysate translation reaction was used to recreate HIV capsid formation in vitro. Production of HIV-1 Pr55Gag and of a matrix-deleted Gag construct resulted in the formation of a subset of Gag protein structures with an equilibrium density of 1.15 g/ml. Gel filtration chromatography revealed these Gag protein structures to be larger than 2 x 10(6) Da, consistent with the formation of large multimers or capsids. These Gag protein structures were protease sensitive in the absence of detergent, indicating that they did not contain a complete lipid envelope. Spherical structures were detected by electron microscopy within the reticulocyte lysate reaction mixtures and appeared essentially identical to immature HIV capsids or retrovirus-like particles. These results demonstrate that the HIV Gag protein is capable of producing immature capsids in a cell-free reaction and that such capsids lack a complete lipid envelope.     

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.     

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.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

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.     

3-O-(3',3'-dimethysuccinyl) betulinic acid inhibits maturation of the human immunodeficiency virus type 1 Gag precursor assembled in vitro

3-O-(3',3'-Dimethysuccinyl) betulinic acid (PA-457) has been shown to potently inhibit human immunodeficiency virus (HIV) replication in culture. In contrast to inhibitors that act upon the viral proteinase, PA-457 appears to block only the final maturational cleavage of p25CA-p2 to p24CA. However, attempts to replicate this effect in vitro using recombinant Gag have failed, leading to the hypothesis that activity is dependent upon the assembly state of Gag. Using a synthesis/assembly system for chimeric HIV type 1 Gag proteins, we have replicated the activity of PA-457 in vitro. The processing of assembled chimeric Gag can be inhibited by the addition of drug with only the final cleavage of p25CA-p2 to p24CA blocked. Consistent with our hypothesis and with previous findings, inhibition appears specific to Gag assembled into an immature capsid-like structure, since synthetic Gag that remains unassembled is properly processed in the presence of the compound. To further analyze the authenticity of the assay, PA-457 was tested in parallel with its inactive parental compound, betulinic acid. Betulinic acid had no effect upon p25 processing in this system. Analysis of a PA-457-resistant mutant, A1V, in this system pointed to more rapid cleavage as a possible mechanism for resistance. However, characterization of additional mutations at the cleavage site and in p2 suggests that resistance does not strictly correlate with the rate of cleavage. With the establishment of an in vitro assay for the detection of PA-457 activity, a more detailed characterization of its mechanism of action will be possible.     

Assembly of human immunodeficiency virus precursor gag proteins

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 A is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.     

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein

The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.