Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene-encoded precursor poly-proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M-PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport-defective mutant-infected cells, suggesting that transport of M-PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein-lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding-defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.     

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.     

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 Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.     

Basic residues in the Mason-Pfizer monkey virus gag matrix domain regulate intracellular trafficking and capsid-membrane interactions

Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.     

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.     

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.     

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.     

Type D Retrovirus Capsid Assembly and Release Are Active Events Requiring ATP

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-d-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Assembly and release of nascent retrovirus particles requires that the viral precursor polyproteins and genomic RNAs, and certain host cell tRNAs, migrate to the plasma membrane, where budding occurs. Two discrete intracellular transport pathways are utilized during the assembly of the infectious virion. The viral glycoproteins are synthesized on membrane-bound polysomes and are transported through the secretory pathway of the cell to the plasma membrane, where they colocalize with the immature capsid during the budding process (20). The major structural proteins of the viral capsid and the enzymatic proteins are synthesized in the cytoplasm on free polysomes and are transported to the underside of the plasma membrane (13, 36). While many of the details of the secretory pathway have been established, the mechanisms for intracytoplasmic protein transport are poorly understood.The major structural polyprotein (Gag) of a nascent retrovirus capsid is encoded by the gag gene. Unlike most enveloped RNA viruses in which the viral glycoproteins mediate assembly by stabilizing the interactions between the capsid proteins and the viral membrane, retroviral Gag proteins can drive capsid assembly and budding in the absence of all the other viral gene products (19, 55, 58). As such, they contain all cis-acting information necessary for intracytoplasmic transport, capsid assembly, membrane binding, envelopment, and release from the cell surface. Assembly of the immature retrovirus capsid begins shortly after the Gag polyproteins are synthesized and modified by myristylation (15, 17, 40, 47–49). The Gag proteins of most retroviruses (the type C avian and mammalian viruses, lentiviruses, and human T-cell leukemia virus/bovine leukemia virus-related viruses) migrate directly to the plasma membrane, where they coalesce into spherical, immature capsids and simultaneously bud through the lipid bilayer, thereby acquiring their envelope. During or shortly after release, the Gag protein is cleaved by the viral protease into the internal structural (NH₂-MA [matrix], CA [capsid], and NC [nucleocapsid]) proteins of the mature, infectious virion (22). In contrast, the Gag proteins of the mammalian and type B and D viruses (mouse mammary tumor virus [MMTV] and Mason-Pfizer monkey virus [M-PMV], respectively) accumulate in the cytoplasm, where they assemble into spherical structures in the absence of membranes. These nascent particles have been referred to as intracytoplasmic type A particles, but by analogy to other viruses and bacteriophages, we have redefined them as procapsids (55). Once assembled, procapsids are transported to the plasma membrane, from which they bud. Despite the different assembly strategies, the processes whereby Gag proteins assemble into procapsids are probably similar since a single amino acid change near the amino terminus of the Gag protein from M-PMV has been shown to convert it to the type C morphogenic pathway (41).Genetic analyses of the gag genes from different retroviruses have shown that Gag proteins contain specific domains which are required for capsid formation. A membrane binding (M) domain has been located at the amino-terminal end of Gag of several retroviruses (31, 43, 60, 61). A late (L) domain functions during the budding and release. In Rous sarcoma virus (RSV) and M-PMV, the L domain is located between the MA and CA domains (57, 59). An equivalent domain in the lentiviruses has been found near the carboxy terminus of the Gag precursor (34). A third domain (I), located near the CA-NC junction, appears to be a region of interaction between Gag proteins (3, 56). Despite the lack of any extensive sequence similarities between different Gag proteins, there is functional conservation between assembly domains. Chimeric Gag proteins containing the M, L, and I domains from different retroviruses can assemble into capsid-like structures and mediate budding at the plasma membrane (3, 9, 10, 34).The M-PMV Gag protein contains additional assembly elements which influence procapsid assembly, stability, and transport. This virus contains a region within Gag (known as p12) that is not found in either the type C viruses or lentiviruses. It has been suggested from biochemical data derived from studies with p12 deletion mutants that this domain assists in assembly by stabilizing intermolecular Gag associations (50). Protein stability and protein/procapsid transport depend on sequences in the MA domain which appear to be distinct from the M domain. As mentioned above, a single point mutation in MA at residue 55 results in a Gag protein that no longer assembles in the cytoplasm but rather assembles at the plasma membrane. This mutation lies within an 18-amino-acid region of the MA domain that has sequence similarity only to the type B retroviruses (41). The nuclear magnetic resonance-derived solution structure of a nonmyristylated M-PMV MA protein indicates that this region folds into a structured turn which is solvent accessible in the monomer and trimer models (8). Moreover, this structural feature is absent in human immunodeficiency virus (HIV), simian immunodeficiency virus, human T-cell leukemia virus, and bovine leukemia virus MA proteins (7, 18, 27–30, 37). It is reasonable, therefore, to suspect that this region contains a cytoplasmic protein transport signal which must interact with a cellular factor. In contrast, other mutations in either the myristic acid addition signal or at a variety of positions elsewhere in the MA coding region result in Gag proteins that fail to be released as virus-like particles despite assembling into procapsids in the cytoplasm (40, 43). Thus, the M-PMV Gag protein appears to contain a second cytoplasmic transport signal which normally directs assembled procapsids and not unassembled Gag proteins to the plasma membrane. It is implied in this model that the M-PMV Gag protein must utilize multiple cellular components during the different stages of assembly and release.The type D retroviruses provide a useful system for studying morphogenic events since procapsid assembly, protein transport, and budding are temporally and spatially unlinked. We report here that in infected cells and an in vitro translation/assembly system, procapsid assembly and transport to the plasma membrane require ATP. Thus, cellular proteins do play an active role during at least two stages of M-PMV morphogenesis.     

Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis.

The major capsid (CA) protein of retroviruses possesses a stretch of 20 amino acids, called the major homology region (MHR), which is evolutionarily conserved and invariant in location within the primary sequence of the protein. The function of this region was investigated by examining the effect of random single-amino-acid substitutions within the central 13 positions of the MHR on the life cycle of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive D-type retrovirus. When these mutants were subcloned into an M-PMV proviral vector and expressed in COS cells, one of two major phenotypes was observed. The first group, containing three mutants bearing drastic amino acid substitutions, was unable to assemble capsids in the cytoplasm of the host cell. The second and more common group of mutants was able to assemble and release virions, but these either displayed greatly reduced levels of infectivity or were completely noninfectious. Included within this second group were two mutants with unusual phenotypes; mutant D158Y exhibited a novel cleavage site for the viral protease that resulted in cleavage of the major capsid protein, p27 (CA), within the MHR, whereas mutant F156L appeared to have lost a major site for antibody recognition within the mature CA protein. The results of this mutagenic analysis suggest that changes in the MHR sequence can interfere with the assembly of viral capsids and block an early stage of the infection cycle of M-PMV.     

A Proline-Rich Motif (PPPY) in the Gag Polyprotein of Mason-Pfizer Monkey Virus Plays a Maturation-Independent Role in Virion Release

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.     

A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus

Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenic process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that prevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed.     

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env -gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.     

The role of the S-S bridge in retroviral protease function and virion maturation

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.     

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.     

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.     

A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules

Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.     

Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane.

The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.