Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.     

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.     

Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena.

Poliovirus infection leads to an increase of phospholipid synthesis and the proliferation of new membranes, giving rise to a great number of cytoplasmic vesicles in the infected cells. Viral RNA replication is physically associated with these newly-synthesized membranes. Cerulenin, an inhibitor of lipid biosynthesis, effectively blocks the growth of poliovirus in HeLa cells. The presence of cerulenin after virus entry prevents the synthesis of poliovirus proteins. However, if this antibiotic is added at later stages of the virus replication cycle, it has no effect on viral translation itself, nor on the proteolytic processing and myristoylation of poliovirus proteins. The synthesis of viral, but not cellular RNA is selectively inhibited by cerulenin. Analysis of the viral RNA made in poliovirus-infected cells by specific minus-or plus-stranded RNA probes suggests a selective blockade by cerulenin of plus-strand RNA synthesis. Finally, the synthesis of phospholipids and the proliferation of membranes does not take place if cerulenin is added to the culture medium. These findings indicate that continuous phospholipid synthesis is required for efficient poliovirus genome replication and provide new insights towards the understanding of the molecular events that occur during poliovirus growth.     

Identification of HTLV-I gag protease and its sequential processing of the gag gene product

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.     

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.     

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Fibronectin is a non-viral substrate for the HIV proteinase

The retrovirus encoded proteinase (PR) is required for the proper maturation of viral particles into infectious virus. The PR had been considered highly substrate specific, cleaving exclusively the viral gag and gag-pol protein precursor. It has recently been reported, however, that cytoskeleton and other cellular filament proteins can be cleaved by the HIV-1 PR. Here we have evidence that a cell-associated protein, the fibronectin (A-chain), is also cleaved in vitro specifically by this PR. The possibility of a cytotoxic role of the PR is conceivable.     

Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates.

Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.     

Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus

We have used a recombinant vaccinia virus (VV) which expresses high levels of human immunodeficiency virus-1 (HIV-1) gag proteins to analyze the processing pathway of the gag p55 precursor. HIV-1 gag proteins were isolated from [3H]leucine-labeled VV:gag-infected H9 T lymphocytes by immunoprecipitation with either anti-p24, anti-p17, or anti-p6 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that processing of the p55 precursor involves three major intermediates (p41a, p41b, and p39). The p41a and p39 proteins contain the p17 and p24 protein segments, and the p41b is comprised of p24 and p15 segments. On two-dimensional gels, each intermediate as well as the mature p24 and p17 proteins migrated as distinct species. [3H]Myristic acid labeling of the HIV-1 gag proteins revealed that in addition to p55 and p17, the p41a and p39 intermediates, but not p41b, are myristylated, confirming that myristylation occurs at the NH2 terminus before cleavage of the p55 precursor protein. We conclude that the myristylated HIV-1 gag p55 precursor is initially cleaved at random either at the p17/p24 junction or at two sites between p24 and p15 proteins, resulting in three intermediates (p41a, p41b, and p39) which are subsequently cleaved to yield mature gag proteins.     

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.     

Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins.

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Development of HIV/AIDS vaccine using chimeric gag-env virus-like particles

We attempted to develop a candidate HIV/AIDS vaccine, by using unprocessed HIV-2 gag pr45 precursor protein. We found that a 45 kDa unprocessed HIV-2 gag precursor protein (pr45), with a deletion of a portion of the viral protease, assembles as virus-like particles (VLP). We mapped the functional domain of HIV-2 gag VLP formation in order to find the minimum length of gag protein to form VLP. A series of deletion mutants was constructed by sequentially removing the C-terminal region of HIV-2 gag precursor protein and expressed truncated genes in Spodoptera frugiperda (SF) cells by infecting recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not affect VLP formation. There is a proline-rich region at the amino acid positions 373 to 377 of HIV-2 gag, and replacement of these proline residues by site-directed mutagenesis completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein, and zinc finger domains, are dispensable for gag VLP assembly, but the presence of at least one of the three prolines at amino acid positions 373, 375 or 377 of HIV-2NIH-Z is required for VLP formation. Animals immunized with these gag particles produced high titer antibodies and Western blot analyses showed that anti-gag pr45 rabbit sera react with p17, p24 and p55 gag proteins of HIV-1. We then constructed chimeric gag genes, which carry the hypervariable V3 region of HIV-1 gp120, because the V3 loop is known to interact with chemokine receptor as a coreceptor, and known to induce the major neutralizing antibodies and stimulate the cytoxic T lymphocyte responses in humans and mice. We expressed chimeric fusion protein of HIV-2 gag with 3 tandem copies of consensus V3 domain that were derived from 245 different isolates of HIV-1. In addition, we also constructed and expressed chimeric fusion protein that contains HIV-2 gag with V3 domains of HIV-1IIIB, HIV-1MN, HIV-1SF2 and HIV-1RF. The chimeric gag-env particles had a spherical morphology, and the size was slightly larger than that of a gag particle. Immunoprecipitation and Western blot analyses show that these chimeric proteins were recognized by HIV-1 positive human sera and antisera raised against V3 peptides, as well as by rabbit anti-gp120 serum. We obtained virus neutralizing antibodies in rabbits by immunizing these gag-env VLPs. In addition, we found that gag-env chimeric VLPs induce a strong CTL activity against V3 peptide-treated target cells. Our results indicate that V3 peptides from all major clades of HIV-1 carried by HIV-2 gag can be used as a potential HIV/AIDS vaccine.     

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.     

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.     

Substitution of proline with pipecolic acid at the scissile bond converts a peptide substrate of HIV proteinase into a selective inhibitor

The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC50 of approximately 1 microM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.     

Recombinant feline herpesviruses expressing feline leukemia virus envelope and gag proteins.

We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp70 and p15E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp70 proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.