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Molecular mechanisms involved in the differential expression of gag gene products by clonal isolates of a primate sarcoma virus.

Clonal isolates of an early passage stock of woolly monkey sarcoma virus (WSV) have been shown to code for different numbers of woolly monkey helper leukemia virus gag gene products. In the present report, the molecular mechanisms responsible for their differential expression of gag gene products have been analyzed. Three WSV RNA genomes were shown to possess sedimentation coefficients consistent with the differences demonstrated in their allotments of helper viral sequences. The WSV variant (WSV clone 9) that expressed no detectable proteins was shown to contain the largest amount of helper viral information. Moreover, there was no additive hybridization of the WLV complementary DNA probe by RNA of this WSV clone and that of a WSV clone coding for several gag gene products. These results suggest that the lack of expression of gag gene products by WSV clone 9 is not due to a major deletion of helper viral gag gene sequences. Similar levels of WLV-specific RNA were demonstrated in cells nonproductively transformed by each WSV clone, arguing that the ability to express gag gene proteins was not related to the magnitude of viral RNA transcription. Taken together, the results are most consistent with a mechanism by which small deletions or point mutations in the genomes of some WSV variants result in premature termination of translation or synthesis of immunologically nonreactive gag gene proteins. The present findings have implications concerning the effects of evolutionary selective pressures on helper viral genetic information in mammalian transforming viruses.

Base Sequence↗

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.

Alpharetrovirus↗

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.

Animals↗

Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus.

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.

Amino Acid Sequence↗

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag.

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.

AIDS Vaccines↗

Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1.

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

Alleles↗

[35S]autoradiographic study of sulfated GAG accumulation and turnover in embryonic mouse tooth germs.

The accumulation of sulfated GAG in embryonic mouse molars before, during, and after terminal differentiation of odontoblasts was localized by [35S]autoradiography combined with the use of chondroitin ABC lyase. Much more sulfated GAG were accumulated in the dental papilla than in the dental epithelium. High incorporation of [35S]sulfate occurred at the epithelio-mesenchymal junction, which is the site of dental basement membrane and predentin. Before terminal differentiation of odontoblasts, the distribution of sulfated GAG was uniform at the basement membrane. After the onset of terminal differentiation of odontoblasts, much more sulfated GAG accumulated at the tip of principal cusps than at the apical (inferior) parts of cusps, and sulfated GAG were then found to be degraded more rapidly at the epithelio-mesenchymal junction than at other parts of the tooth germ. Thus regional variation in the rate of degradation of GAG exists in the tooth germs. Trypsin-isolated dental epithelia cultured in vitro synthesized a new basement membrane that could be labeled with [3H]glucosamine but not with 35SO4(-2). The epithelial-derived basal lamina contains little or no sulfatated GAG.

Animals↗

Membrane relocation but not tight binding of human immunodeficiency virus type 1 Gag particles myristoylated in Escherichia coli.

Expression of human immunodeficiency virus Gag protein and the N-terminal matrix (MA) domain in Escherichia coli yielded spherical structures in the cytoplasm. When human N-myristoyltransferase was coexpressed, both Gag and MA were fully myristoylated and spherical structures were relocated in close proximity to the cytoplasmic membrane. However, neither myristoylated Gag nor MA exhibited tight binding to E. coli membrane, suggesting that myristoylation in E. coli did not confer membrane affinity on Gag despite the relocation. Our data also suggest that the morphogenetic pathway of Gag particles in prokaryotic cells differs from that in eukaryotic cells despite biochemical similarities of in the form of Gag expressed.

Acyltransferases↗

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

AIDS Vaccines↗

Determination of gag and env subtypes of HIV-1 detected among injecting drug users (IDUs) in Manipur, India: evidence for intersubtype recombination.

The majority of HIV-1 transmission in Manipur, one of the northeastern states of India, is through the sharing of needles and syringes among the injecting drug users (IDUs). A total of 28 HIV seropositive samples were used to determine the HIV-1 subtypes with respect to both gag and envelope genes. The specific regions within gag and envelope genes were amplified from PBMC DNA by nested PCR using appropriate primers. These amplicons were used in heteroduplex mobility assay followed by DNA sequencing. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 89% of samples (25/28) were infected with subtype C with respect to both gag and envelope genes; however, 11% of the samples (3/28) showed subtype discordance with respect to the envelope (C2-V3) and gag (p24-p7) genomic regions. Simplot analysis of the discordant samples showed the presence of intersubtype recombination between subtype C and Thai B; two samples were found to be subtype C in envelope but Thai B in gag, whereas, one sample was found to be subtype Thai B in envelope and 'C' in gag region.

Adult↗

Preferential binding of poly(A)-binding protein 1 to an inhibitory RNA element in the human immunodeficiency virus type 1 gag mRNA.

Human immunodeficiency virus type 1 (HIV-1) mRNAs encoding structural proteins contain multiple inhibitory/instability elements (INS), which decrease the efficiency of viral protein expression. We have previously identified a strong INS element (INS-1) within the p17(gag) coding region. Here we show that poly(A)-binding protein 1 (PABP1) binds preferentially to INS-1 within the p17(gag) mRNA, but not to a mutated mRNA in which INS-1 function is eliminated. Competition experiments performed in the presence of different nucleic acids and homoribopolymers demonstrated preferential binding of PABP1 to the INS-1-containing RNA. In contrast to HeLa cells and several lymphoid cell lines, certain human glioma cell lines exhibit high levels of gag expression in the absence of Rev upon transient transfection with wild type gag expression vectors. We analyzed extracts of different cell lines and found that the binding of PABP1 to INS-1 RNA is significantly diminished in glial cell extracts. The expression levels of gag correlate with the absence of binding of PABP1 to the INS-1 RNA in cellular extracts. These results suggest a role for PABP1 in the inhibition of gag expression mediated through INS-1.

Blotting, Western↗

Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities.

The presence of cytotoxic T lymphocytes (CTL) to the gag antigens of human immunodeficiency virus (HIV) has been described in infected populations. We found that the majority of this immune response as measured in bulk CTL assays of unstimulated peripheral blood mononuclear cells (PBMC) is directed against the p24 component of the p55 gag precursor protein. Using limiting dilution analysis of this effector cell population we confirm that the majority of activated gag-specific CTL circulating in the PBMC of infected hemophilic patients are directed at p24 determinants and are present at frequencies of 1/36,000 to 1/86,000 lymphocytes. By performing in vitro stimulation after limiting dilution, the precursor population of gag-specific CTL are characterized and quantitated. HIV gag-specific CTL precursors are identified at frequencies of 1/1700 to 1/17,000 lymphocytes and are made up of cells with both p17 and p24 specificities. No HIV gag-specific CTL precursor cells are identified in the PBMC of HIV-uninfected individuals. These studies demonstrate that CTL directed at both p17 and p24 determinants make up the cellular immune repertoire in HIV-infected individuals but that only the p24-specific CTL are routinely found in an activated state in the circulation.

CD8 Antigens↗

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins.

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be approximately 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

Capsid Proteins↗

Overproduction, purification, and diagnostic use of the recombinant HIV-1 Gag proteins, the precursor protein p55 and the processed products p17, p24, and p15.

HIV-1 Gag protein precursor p55, and its processed products, p17, p24, and p15 were overproduced in Escherichia coli and purified to near homogeneity. To study the antigenic properties and the potentiality as the diagnostic and prognostic reagents, varying amounts of the purified Gag proteins were dotted onto the polyvinylidene difluoride membrane and reacted with 40 sera of HIV-1-infected individuals (35 AC, 1 ARC, and 4 AIDS patients) and 10 sera of normal healthy donors. p55 reacted with 40 (100%) sera of HIV-1 carriers, while p17, p24, and p15 reacted with 37 (92.5%), 35 (87.5%) and 34 (85%) of the 40 sera of HIV-1 carriers, respectively. On the whole, the reaction of p55 was especially strong and that of p15 was the weakest. p55 showed the strongest reaction among the four Gag proteins with all specimens, and it showed a positive reaction with a carrier serum with which none of the processed Gag proteins showed a positive reaction. Therefore, p55 is the most useful antigen among the four Gag proteins for detection of the Gag antibodies and may even be one of the most useful antigens for the diagnosis of HIV-1 infection.

Chromatography, Gel↗

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

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

Capsid Proteins↗

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.

Amino Acid Sequence↗

Protection against Friend retrovirus-induced leukemia by recombinant vaccinia viruses expressing the gag gene.

High sequence variability in the envelope gene of human immunodeficiency virus has provoked interest in nonenvelope antigens as potential immunogens against retrovirus infection. However, the role of core protein antigens encoded by the gag gene in protective immunity against retroviruses is unclear. By using recombinant vaccinia viruses expressing the Friend murine leukemia helper virus (F-MuLV) gag gene, we could prime CD4+ T-helper cells and protectively immunize susceptible strains of mice against Friend retrovirus infection. Recovery from leukemic splenomegaly developed more slowly after immunization with vaccinia virus-F-MuLV gag than with vaccinia virus-F-MuLV env; however, genetic nonresponders to the envelope protein could be partially protected with Gag vaccines. Class switching of F-MuLV-neutralizing antibodies from immunoglobulin M to immunoglobulin G after challenge with Friend virus complex was facilitated in mice immunized with the Gag antigen. Sequential deletion of the gag gene revealed that the major protective epitope was located on the N-terminal hydrophobic protein p15.

Amino Acid Sequence↗

Genetic assay for multimerization of retroviral gag polyproteins.

We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and O. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.

Cloning, Molecular↗