Monitoring processed,mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen

Protease inhibitors (PIs) block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55^gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.     

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7-p1-p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7-p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1-p6) and tertiary (p7-p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7-p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.     

The SIVmac239 Pr55Gag isoform, SIV p43, suppresses proteolytic cleavage of Pr55Gag

In human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) the gag gene encodes the precursor polyprotein Pr55Gag, which is cleaved by the viral protease to produce the major structural proteins. Recently, it has been shown that HIV and SIV gag RNAs contain internal ribosome entry sites (IRESs) that mediate translation of Pr55Gag [Pr57Gag in HIV type 2 (HIV-2)] isoforms. Previously, we demonstrated that SIVmac239 p43(-), a mutant that does not express the Pr55Gag isoform, SIV p43, replicates more efficiently than wild-type (WT) SIVmac239 in cell culture. In this study, we characterize SIVmac239 p43(-) virion production and demonstrate that, in the absence of SIV p43, cleavage of Pr55Gag is increased in budded virions, resulting in a higher percentage of mature particles. Additionally, intracellular cleavage of Pr55Gag is increased in SIVmac239 p43(-), suggesting that SIV p43 suppresses premature cleavage of Pr55Gag by the viral protease.     

Cell-free synthesis of Rauscher murine leukemia virus "gag" and "env" gene products from separate cellular mRNA species.

RNA from cells infected with Rauscher murine leukemia virus (R-MuLV) has been translated in an mRNA-dependent cell-free protein synthesizing system. It was found that a cellular RNA species of about 35 S in size codes for polypeptides of approximately 65,000 MW (Pr65^gag) and 200,000 MW (Pr200^gag) which are immunoprecipitable with antisera directed against the R-MuLV gag proteins p30, p15, p12, and p10. The methionine-containing-tryptic peptides of the 65,000 MW polypeptide translated from cellular 35 S RNA were identical to those of authentic Pr65^gag. Translation of RNA in the 25–35 S size class suggests that while Pr65^gag can be translated by RNA throughout this size range, Pr200^gag-pol translation is restricted to mRNA which sediments at 35 S. Antiserum directed against the R-MuLV envelope protein gp69/71 recognized a polypeptide of 68,000 MW, designated Pr68^env, which was coded for by RNA which sedimented at about 22 S in sucrose gradients and which had a minimum size of about 1.25 × 10⁶ daltons as estimated by agarose gel electrophoresis. Tryptic maps of Pr68^env showed it to contain all of the methionine-labeled tryptic peptides and most of the tyrosine-containing tryptic peptides characteristic of gPr90^env the authentic R-MuLV glycosylated envelope precursor.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

The NH2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76gag).

Pr76^gag, the polyprotein precursor to avian oncovirus internal structural proteins (gag proteins) was isolated by immunoprecipitation from a mRNA-dependent in vitro translation system programmed with genomic (35 S) RNA purified from the Prague strain of Rous sarcoma virus (PR-RSV-C). Attempts to sequence Pr76 were initially unsuccessful due to the presence of a blocked NH₂-terminus. However, when virion 35 S RNA was translated under conditions that prevent NH₂-terminal acetylation, the sequence: Met-Glu-Ala-Val-Ile-Lys-Val-Ile-X-X-Ala-X-Lys was obtained by automated Edman degradation. Since the NH₂-terminal methionine is derived from Met-tRNA_f^Met we conclude that this sequence repesents a primary translation product. Sequence analysis of the supernatant remaining after immunoprecipitation suggests that no viral-related proteins are synthesized which are not precipitated by anti-gag serum. Previous studies employing pactamycin mapping of viral proteins synthesized in infected chick cells indicated that the virion protein p19 was located close to the NH₂-terminus of Pr76^gag (Vogt, et al., J. Mol. Biol.96, 471–493, 1975). We therefore prepared tryptic peptides of p19 purified from virions, separated them by cation exchange chromatography, and determined the amino acid composition of the 19a and 19d peptides. The amino acid composition of the 19a, but not the 19d, peptide correlated with the first six amino acids determined by sequencing. In addition, 19a was the onyl p19-derived peptide not susceptible to digestion by leucine amino peptidase, indicating the presence of a blocked NH₂-terminus. We conclude that the NH₂-terminal sequence of Pr76^gag represents the NH₂-terminus of p19. Comparison of the amino acid sequence with the previously published nucleotide sequence of the 5′ end of 35S RNA from PR-RSV-C (Haseltine et al., Proc. Nat. Acad. Sci. USA74, 989–993, 1977; Shine et al., Proc. Nat. Acad. Sci. USA74, 1473–1477, 1977) shows that synthesis of Pr76^gag is not initiated within the first 119 nucleotides of the viral genome.     

Effects of gag mutations on human immunodeficiency virus type 1 particle assembly,processing, and cyclophilin A incorporation

A series of human immunodeficiency virus (HIV) mutants was constructed either by deletion or by linker insertion at various regions in the gag coding sequences. The ability of each mutant to assemble virus particles and to process them proteolytically, as well as incorporate cyclophilin A, was analyzed by Western immunoblot. This investigation indicated that most of the gag mutants were assembled and released at a level comparable to that of wild-type virus. In an assay involving a single cycle of infection, mutants containing significant levels of cyclophilin A showed less in trans interference effects on wild-type infectivity than did cyclophilin A-deficient mutants. Mutations in the N-terminal two-thirds of capsid protein severely disrupted cyclophilin A incorporation, but they affected virus processing only slightly to moderately. Virions released from cyclosporine-treated cells were processed, as well as virions made by the mock-treated cells. Also, protease inhibitor treatment had no detectable effect on the cyclophilin A incorporation. These results indicate that cyclophilin A incorporation is not required for virus particle processing and that virus processing does not affect cyclophilin A incorporation.     

An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA.

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA (M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins (gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) (R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors (K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.     

p65 of Gazdar murine sarcoma viruses contains antigenic determinants from all four of the murine leukemia virus (MuLV) gag polypeptides (p15, p12, p30, and p10) and can be cleaved in vitro by the MuLV proteolytic activity

Thin-section electron micrographs of Gazdar murine sarcoma virus (Gz-MSV) particles showed that 100% of the particles possessed an immature morphology. Correspondingly, p65 (the major 65,000-dalton protein observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for Gz-MSV particles) possessed antigenic determinants from all four of the murine leukemia virus (MuLV) Pr65^gag polypeptides—that is, p30, p15, p12, and p10. This result is in contrast to earlier observations (A. Pinter and E. deHarven (1979), Virology, 99, 103–110) which reported that p65 lacked antigenic determinants of MuLV p10. It is consistent with the recent finding of Maxwell and Arlinghaus ((1981), J. Virol., 39, 963–967) that Gz-MSV p65, when cleaved in vitro, gives rise to a polypeptide with the size and antigenic determinant of MuLV p10. Thus, we suggest that Gz-MSV p65 should be designated as Gz-MSV Pr65^gag. We also found that Gz-MSV Pr65^gag could be cleaved in vitro by using a partially purified proteolytic factor that had been derived from Moloney murine leukemia virus (M-MuLV) by Sephadex G-75 column chromatography (Y. Yoshinaka and R. B. Luftig (1980), J. Gen. Virol., 48, 329–340). Protein bands were produced that migrated on gels and had the same antigenic determinants as the MuLV intermediates Pr40^gag (p30, p10) and Pr27^gag (p15, p12). Pr55^gag (p15, p12, p30), a minor component, was also produced. Additional incubation of Gz-MSV Pr65^gag led to a breakdown of the intermediate polyproteins into the four MuLV gag polypeptides p30, p10, p15, and p12. The final processing of Pr55^gag and Pr40^gag occurred more rapidly than that of Pr27^gag. It thus seems that in vitro sequentially different processing events are involved in production of the four internal gag antigens from Gz-MSV Pr65^gag.     

Cleavage and purification of prokaryotically expressed HIV gag and env fusion proteins for detection of HIV antibodies in the ELISA

Parts of the gag p24 and the gp41 transmembrane protein of the human immunodeficiency virus HIV-1 were expressed as fusion proteins in Escherichia coli, using an expression vector carrying aa 1-375 of the lac-Z gene linked to the recognition sequence for the blood coagulation factor Xa. Fusion proteins were cleaved into the bacterial and viral portion and the viral polypeptide was purified by a molecular sieve column. The purified viral antigens were tested with 288 human sera in the enzyme-linked immunosorbent assay (ELISA) technique. Comparison with commercially available tests showed comparable sensitivity and a higher specificity of the gag/env-ELISA for borderline reactive sera.     

The critical role of proximal gag sequences in feline immunodeficiency virus genome encapsidation

Retroviral RNA encapsidation is mediated by specific interactions between viral Gag proteins and cis-acting packaging sequences in genomic RNA. Feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be discrete and noncontinuous, comprising one region at the 5' end of the genomic mRNA (R-U5) and another region that mapped within the proximal 311 nt of gag. To aid comparative understanding of lentiviral encapsidation and refinement of FIV vector systems, we used RNase protection assays (RPAs) of cellular and virion RNAs to investigate in detail the gag element. mRNAs of subgenomic vectors as well as of full-length molecular clones were optimally packaged into viral particles and resulted in high-titer FIV vectors when they contained only the proximal 230 nucleotides (nt) of gag. Further 3' truncations of gag sequences progressively diminished encapsidation and transduction. Deletion of the initial ninety 5' nt of the gag gene abolished mRNA packaging, demonstrating that this segment is indispensable for encapsidation. Focusing further on this proximal sequence, we found that a deletion of only 13 nt at the 5' end of gag impaired encapsidation of subgenomic vector and proviral RNAs.     

Comparative humoral responses to human immunodeficiency virus type 1-p24gag linear B-cell epitopes among individuals showing atypical western immunoblotting reactions and implications for diagnosis.

Serum specimens from 25 individuals with an isolated human immunodeficiency virus type 1 (HIV-1) core antigen reactivity in a Western immunoblot test were examined for their reactivities with HIV-1 virions, control cellular antigens, HIV-1-Bru p24gag recombinant protein (p24gag), and a panel of 22 p24gag-derived peptides. The results were as follows: (i) serum specimens from eight HIV-1-uninfected subjects did bind to virions but failed to bind to p24gag; (ii) sera from 13 HIV-1-uninfected subjects and from one HIV-2-infected patient reacted with HIV-1 virions and p24gag but failed to bind to any of the peptides expressing major p24gag epitopes, and (iii) 3 serum specimens obtained from one neonate carrying anti-HIV-1 maternal antibody and from two HIV-1-infected subjects who had seroconverted during the study reacted with HIV-1 virions, p24gag, and one or more peptides containing the major p24gag epitopes. Our data suggest that the combination of p24gag and appropriate peptides could be useful for resolution when atypical Western immunoblot results are encountered.     

Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti-gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.