The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.     

The murine endogenous retrovirus MIA14 encodes an active aspartic proteinase that is functionally similar to proteinases from D-type retroviruses

Murine intracisternal A-type particles (IAPs) are endogenous retroviruses showing sequence homologies to B/D- and avian C-type retroviruses and a gene expression strategy similar to that of D-type retroviruses. These viruses form immature particles in the endoplasmic reticulum and do not release extracellular virions, but are competent for retrotransposition within the virus-producing cell. It had been assumed that lack of polyprotein processing and maturation is due to a defect in the viral proteinase (PR), but recent experiments have shown that polyprotein processing occurs when assembly of the mouse IAP MIA14 is artificially directed to the plasma membrane. We have expressed and purified recombinant MIA14 PR and show that it undergoes N- and C-terminal autoprocessing at defined sites. Using peptide cleavage and inhibition assays and in vitro cleavage of recombinant HIV-1 and MIA14 Gag polyproteins, we show that MIA14 PR is a catalytically competent enzyme comparable in its efficiency to PRs from type D exogenous retroviruses. MIA14 PR is related to the PR of Mason-Pfizer monkey virus both functionally and with respect to its expression strategy, and is distinct from HIV-1 PR with respect to substrate specificity and catalytic efficiency. These findings reveal a functional and possibly evolutionary relationship between MIA14 and D-type retroviruses and imply that a functional PR may be relevant for intracellular retrotransposition even in the case of an endogenous retrovirus that does not produce extracellular virus.     

Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation.

The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Kr?usslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959.     

Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.     

An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.

Infectious retrovirus particles are derived from structural polyproteins which are cleaved by the viral proteinase (PR) during virion morphogenesis. Besides cleaving viral polyproteins, which is essential for infectivity, PR of human immunodeficiency virus (HIV) also cleaves cellular proteins and PR expression causes a pronounced cytotoxic effect. Retroviral PRs are aspartic proteases and contain two copies of the triplet Asp-Thr-Gly in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural role. We have changed this threonine in HIV type 1 PR to a serine. The purified mutant enzyme had an approximately 5- to 10-fold lower activity against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial expression and yielded significantly reduced cleavage of cytoskeletal proteins in vitro. Cleavage of vimentin in mutant-infected T-cell lines was also markedly reduced. Mutant virus did, however, elicit productive infection of several T-cell lines and of primary human lymphocytes with no significant difference in polyprotein cleavage and with similar infection kinetics and titer compared with wild-type virus. The discrepancy between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an increase in Km which may not be relevant at the high substrate concentration in the virus particle. This mutation enables us therefore to dissociate the essential function of PR in viral maturation from its cytotoxic effect.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

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.     

Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers.

Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors.     

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.     

Transport and processing of the Rous sarcoma virus Gag protein in the endoplasmic reticulum.

The Gag proteins of replication-competent retroviruses direct budding at the plasma membrane and are cleaved by the viral protease (PR) just before or very soon after particle release. In contrast, defective retroviruses that bud into the endoplasmic reticulum (ER) have been found, and morphologically these appear to contain uncleaved Gag proteins. From this, it has been proposed that activation of PR may depend upon a host factor found only at the plasma membrane. However, if Gag proteins were cleaved by PR before the particle could pinch off the ER membrane, then the only particles that would remain visible are those that packaged smaller-than-normal amounts of PR, and these would have an immature morphology. To distinguish between these two hypotheses, we made use of the Rous sarcoma virus (RSV) Gag protein, the PR of RSV IS included on each Gag molecule. To target Gag to the ER, a signal peptide was installed at its amino terminus in place of the plasma membrane-binding domain. An intervening, hydrophobic, transmembrane anchor was included to keep Gag extended into the cytoplasm. We found that PR-mediated processing occurred, although the cleavage products were rapidly degraded. When the anchor was removed, allowing the entire protein to be inserted into the lumen of the ER, Gag processing occurred with a high level of efficiency, and the cleavage products were quite stable. Thus, PR activation does not require targeting of Gag molecules to the plasma membrane. Unexpectedly, molecules lacking the transmembrane anchor were rapidly secreted from the cell in a nonmembrane-enclosed form and in a manner that was very sensitive to brefeldin A and monensin. In contrast, the wild-type RSV and Moloney murine leukemia virus Gag proteins were completely insensitive to these inhibitors, suggesting that the normal mechanism of transport to the plasma membrane does not require interactions with the secretory pathway.     

Spleen necrosis virus gag polyprotein is necessary for particle assembly and release but not for proteolytic processing.

The nature of spleen necrosis virus pol gene expression and the role of gag and gag-pol polyproteins in virion assembly was investigated. The DNA sequence of the gag-pol junction revealed that the two genes occupy the same open reading frame but are separated by an in-frame amber stop codon. Biochemical analysis of gag-pol translational readthrough in vitro and in Escherichia coli suggests that, in a manner similar to that in other mammalian type C retroviruses, amber stop codon suppression is required for pol gene expression. Removal of the gag stop codon had little or no effect on synthesis or cleavage of the polyprotein but interrupted particle assembly. This block could be overcome by complementation with wild-type gag protein.     

The Molecular Architecture of HIV

Assembly of human immunodeficiency virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an immature virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the virus assembly site, the immature virus, the maturation intermediates and the mature virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.     

Effects of blocking individual maturation cleavages in murine leukemia virus gag

A single protein, termed Gag, is responsible for retrovirus particle assembly. After the assembled virion is released from the cell, Gag is cleaved at several sites by the viral protease (PR). The cleavages catalyzed by PR bring about a wide variety of physical changes in the particle, collectively termed maturation, and convert the particle into an infectious virion. In murine leukemia virus (MLV) maturation, Gag is cleaved at three sites, resulting in formation of the matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. We introduced mutations into MLV that inhibited cleavage at individual sites in Gag. All mutants had lost the intensely staining ring characteristic of immature particles; thus, no single cleavage event is required for this feature of maturation. Mutant virions in which MA was not cleaved from p12 were still infectious, with a specific infectivity only approximately 10-fold below that of the wild type. Particles in which p12 and CA could not be separated from each other were noninfectious and lacked a well-delineated core despite the presence of dense material in their interiors. In both of these mutants, the dimeric viral RNA had undergone the stabilization normally associated with maturation, suggesting that this change may depend upon the separation of CA from NC. Alteration of the C-terminal end of CA blocked CA-NC cleavage but also reduced the efficiency of particle formation and, in some cases, severely disrupted the ability of Gag to assemble into regular structures. This observation highlights the critical role of this region of Gag in assembly.     

An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.     

Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

Avian retroviruses (with the notable exception of spleen necrosis virus) express their protease (PR) both in their gag and their gag-pol polyprotein precursors, in contrast to other retroviruses, notably, the mammalian retroviruses, in which PR is encoded in the gag-pol polyprotein or in a separate reading frame as a gag-pro product. The consequence is that the avian PR is expressed in stoichiometric rather than catalytic amounts. To investigate the significance of the particular genome organization of the avian retrovirus prototype Rous sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol polyproteins by expressing them from two different plasmids in transfected cells. By using this assay, we showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activation when coexpressed with a protease-deficient gag protein and that the PR domain has a role in viral particle assembly. Furthermore, this complementation assay can be used to investigate the role of the gag domain in the gag-pol polyprotein by determining whether it can rescue a defect in the gag polyprotein. We report here the results of such an experiment, which studied a mutation in the N terminus of the gag gene.     

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.     

Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles

The viral genomes of alpha- and gamma-retroviruses follow an outbound route through the cytoplasm before assembling with the budding particle at the plasma membrane. We show here that murine leukemia virus (MLV) RNAs are transported on lysosomes and transferrin-positive endosomes. Transport on transferrin-positive vesicles requires both Gag and Env polyproteins. In the presence of Env, Gag is rerouted from lysosomes to transferrin-positive endosomes, and virion production becomes highly sensitive to drugs poisoning vesicular and endosomal traffic. Vesicular transport of the RNA does not require prior endocytosis, indicating that it is recruited directly from the cytosol. Viral prebudding complexes containing Env, Gag, and retroviral RNAs are thus formed on endosomes, and subsequently routed to the plasma membrane. This may allow retroviruses to hijack the endosomal machinery as part of their biosynthetic pathway. More generally, tethering to vesicles may provide an efficient mechanism for directed RNA transport.