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PubMed2006

Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing.

The proteolytic processing of human immunodeficiency virus (HIV) particles mediated by the viral pol-encoded protease (PR) is essential for viral infectivity. The pol coding sequence partially overlaps with the gag coding sequence and is translated as a Gag-Pol polyprotein precursor. Within Gag-Pol, the C-terminal p6(gag) domain is replaced by a transframe peptide referred to as p6*, which separates the Gag nucleocapsid domain from PR. Several previous in vitro studies have ascribed a PR-suppression regulatory function to p6*. Here, it was demonstrated that an HIV-1 Gag-Pol lacking p6* is efficiently incorporated into virions when coexpressed with HIV-1 Gag precursor. However, the released virions are not processed appropriately and show a greatly reduced viral infectivity. This suggests that the p6* is indispensable during the process of PR-mediated virus particle maturation.

Amino Acid Sequence↗
PubMed1982

Association of gag-myc proteins from avian myelocytomatosis virus wild-type and mutants with chromatin.

The localization of the transformation-specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild-type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A , Q10C , and Q10H , with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation-specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N-terminal region of the polyprotein, showed that the gag-myc proteins in cells transformed by the wild-type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag-erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag-myc proteins of wild-type as well as of mutant origin were found in the nucleus, while 90% of the gag-erb A protein was present in the cytoplasm. Also, pulse-chase analysis indicated that the gag-myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild-type and also mutant gag-myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag-myc protein from MC29-transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.

Animals↗
PubMed1996

The gag coding region of the Drosophila telomeric retrotransposon, HeT-A, has an internal frame shift and a length polymorphic region.

A major component of Drosophila telomeres is the retrotransposon HeT-A, which is clearly related to other retrotransposons and retroviruses. This retrotransposon is distinguished by its exclusively telomeric location, and by the fact that, unlike other retrotransposons, it does not encode its own reverse transcriptase. HeT-A coding sequences diverge significantly, even between elements within the same genome. Such rapid divergence has been noted previously in studies of gag genes from other retroelements. Sequence comparisons indicate that the entire HeT-A coding region codes for gag protein, with regions of similarity to other insect retrotransposon gag proteins found throughout the open reading frame (ORF). Similarity is most striking in the zinc knuckle region, a region characteristic of gag genes of most replication-competent retroelements. We identify a subgroup of insect non-LTR retrotransposons with three zinc knuckles of the form: (1) CX2CX4HX4C, (2) CX2CX3HX4C, (3) CX2CX3HX6C. The first and third knuckles are invariant, but the second shows some differences between members of this subgroup. This subgroup includes HeT-A and a second Drosophila telomeric retrotransposon, TART. Unlike other gag regions, HeT-A requires a -1 frameshift for complete translation. Such frameshifts are common between the gag and pol sequences of retroviruses but have not before been seen within a gag sequence. The frameshift allows HeT-A to encode two polypeptides; this mechanism may substitute for the post-translational cleavage that creates multiple gag polypeptides in retroviruses. D. melanogaster HeT-A coding sequences have a polymorphic region with insertions/deletions of 1-31 codons and many nucleotide changes. None of these changes interrupt the open reading frame, arguing that only elements with translatable ORFs can be incorporated into the chromosomes. Perhaps HeT-A translation products act in cis to target the RNA to chromosome ends.

Amino Acid Sequence↗
PubMed1995

Ribosomal frameshifting at the Gag-Pol junction in avian leukemia sarcoma virus forms a novel cleavage site.

The Gag and Gag-Pol precursors of avian sarcoma leukemia virus (ASLV) are translated from viral genomic-size mRNA at a molar ratio of about 20:1. Translation of Gag is terminated at the stop codon UAG located at the carboxyl-terminus of the viral protease (PR), whereas a ribosomal frameshift occurring at the carboxyl-terminus of Gag allows translation of the Gag-Pol precursor. To determine how PR is released from the Gag-Pol precursor, a single base (A or T) was inserted at the Gag-Pol junction in order to adjust the translation into a single reading frame. These mutations allow processing of the viral precursor when expressed in bacterial cells, but cause cessation of viral production after transfection of avian cells. The viral PR released from the large precursor is one amino acid longer than PR cleaved from the Gag polyprotein and is terminated by an Ile instead of a Leu residue.

Alpharetrovirus↗
PubMed1984

The gag-mos hybrid protein of ts110 Moloney murine sarcoma virus: variation of gene expression with temperature.

A NRK cell clone (6m2 cells) infected with ts110 Moloney murine sarcoma virus (MuSV) produce a gag-mos protein, P85gag-mos, and a truncated gag protein of Mr 58,000d termed P58gag. The gag-mos protein is produced from a 3.5-kb mRNA whereas the gag protein is made from a 4.0-kb mRNA. It has been proposed that the 3.5-kb RNA is produced from the 4.0-kb RNA by a splicing mechanism (R. P. Junghans, E. C. Murphy, Jr., and R. B. Arlinghaus (1982) J. Mol. Biol. 161, 229-255). The results presented here provide further support for this model. The expression of the 3.5-kb RNA and the gag-mos protein increased as the temperature at which 6m2 cells were maintained was lowered from 39 to 28 degrees. This increase coincided with a decrease in both the 4.0-kb RNA and its product P58gag. The optimum temperature for syntheses of both the gag-mos mRNA and its protein was found to be 28 degrees. Consistent with the increase in the level of the gag-mos protein is the increase in the protein kinase activity associated with P85gag-mos and the degree of morphological transformation of 6m2 cells. Thus, the level of P85gag-mos within 6m2 cells is directly proportional to the degree of cell transformation and the amount of the kinase activity associated with the gag-mos protein, providing convincing evidence that P85gag-mos plays a direct role in the neoplastic transformation of these cells.

Animals↗
PubMed2006

Intradermal and oral immunization with recombinant Mycobacterium bovis BCG expressing the simian immunodeficiency virus Gag protein induces long-lasting, antigen-specific immune responses in guinea pigs.

To develop a new recombinant BCG (rBCG) vaccine, we constructed rBCG that expresses the full-length Gag protein of simian immunodeficiency virus (rBCG-SIVGag) at a level of 0.5 ng/mg after 3 weeks of bacterial cell culture. Intradermal (i.d.) inoculation of guinea pigs with 0.1 mg of rBCG-SIVGag resulted in the induction of delayed-type hypersensitivity (DTH) responses to both purified protein derivative (PPD) of tuberculin and SIV Gag p27 protein; responses that were maintained for the duration of the 50-week study. In contrast, guinea pigs orally vaccinated with 160 mg of the same antigen exhibited a long-lasting DTH response to the SIV Gag p27 protein, but mounted no response to PPD. Proliferative responses to SIV Gag p27 and PPD antigens were detected in both i.d. and orally immunized animals; however, the levels of PPD-specific responses were significantly higher in guinea pigs immunized by the i.d. than the oral route. A significant increase in the level of PPD- and SIV Gag p27-specific IFNgamma mRNA expression was also detected in both immunization groups receiving rBCG-SIVGag. In addition, both i.d. and oral immunization with rBCG-SIVGag induced PPD- and SIV Gag p27-specific serum IgG responses. Insertion of the SIV gag gene into BCG did not appear to change the ability of rBCG-immunized animals to elicit PPD-specific immune responses. These results indicate that rBCG-SIVGag has the ability to effectively induce long-lasting, cell-mediated and humoral immunity against both viral and bacterial antigens in guinea pigs, suggesting that rBCG-Gag has the potential to elicit immunities specific not only for tuberculosis but also for HIV at human doses.

Administration, Oral↗
PubMed2004

In vivo homodimerisation of HTLV-1 Gag and MA gives clues to the retroviral capsid and TM envelope protein arrangement.

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.

Amino Acid Sequence↗
PubMed2005

Localization of self-interacting domains within betaretrovirus Gag polyproteins.

The Betaretrovirus genus is characterized by the ability to preassemble immature capsids within the cytoplasm. For Mason-Pfizer monkey virus (M-PMV) this ability depends in part upon the unique Internal Scaffold Domain (ISD) within the p12 region of Gag. In this study, we have further characterized the ability of M-PMV p12 to promote Gag-Gag interaction and have examined the Gag polyprotein of the related mouse mammary tumor virus (MMTV) to potentially identify a region with equivalent function. Using the yeast two-hybrid system, we confirmed that both Gag polyproteins strongly interact, primarily through the CA-NC regions, but also through additional domains N-terminal to CA. For M-PMV, this auxiliary interaction domain was p12. For MMTV, no single strongly self-interacting protein was identified. Instead, MMTV Gag appears to utilize the weak contributions of several protein domains to support the main interaction of its CA-NC. Our findings suggest that, in addition to the canonical NC "I-domain" interaction, MMTV Gag self-association results from the concerted action of multiple regions of the polyprotein while M-PMV Gag relies mainly on its p12 domain.

Amino Acid Sequence↗
PubMed2006

An internal ribosome entry site promotes translation of a novel SIV Pr55(Gag) isoform.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

Animals↗
PubMed2003

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles.

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types.

Animals↗
PubMed1998

HIV-1 Gag binds specifically to RNA stem-loops in the 5' leader sequence.

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro, with a Kd of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325-362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5' of nucleotide 335 require the presence of additional 3' sequences.

Amino Acid Sequence↗
PubMed1998

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Animals↗
PubMed1987

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Amino Acid Sequence↗
PubMed1996

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20 degrees C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

Antiviral Agents↗
PubMed2002

Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends.

Drosophila telomeres are formed by two non-LTR retrotransposons, HeT-A and TART, which transpose only to chromosome ends. Successive transpositions of these telomeric elements yield arrays that are functionally equivalent to the arrays generated by telomerase in other organisms. In contrast, other Drosophila non-LTR retrotransposons transpose widely through gene-rich regions, but not to ends. The two telomeric elements encode very similar Gag proteins, suggesting that Gag may be involved in their unique targeting to chromosome ends. To test the intrinsic potential of these Gag proteins for targeting, we tagged the coding sequences with sequence of GFP and expressed the constructs in transiently transfected Drosophila-cultured cells. Gag proteins from both elements are efficiently transported into the nucleus where the protein from one element, HeT-A, forms structures associated with chromosome ends in interphase nuclei. Gag from the second element, TART, moves into telomere-associated structures only when coexpressed with HeT-A Gag. The results suggest that these Gag proteins are capable of delivering the retrotransposons to telomeres, although TART requires assistance from HeT-A. They also imply a symbiotic relationship between the two elements, with HeT-A Gag directing the telomere-specific targeting of the elements, whereas TART provides reverse transcriptase for transposition.

Animals↗
PubMed2001

HIV type 1 Gag and nucleocapsid proteins: cytoskeletal localization and effects on cell motility.

Cell motility is likely to play a pivotal role in HIV infection by promoting the dissemination of infected cells. On the basis of observations indicating an interaction between HIV-1 Gag and target cell filamentous actin, we hypothesized that these interactions would promote cell motility of HIV-infected cells. Indeed, we have found that HIV-1 infection enhances the chemotactic response of macrophages. To specifically investigate the significance of the interactions between Gag and cellular actin, we transfected NIH 3T3 fibroblasts and HeLa cells with a construct that permits the expression of HIV-1 Gag in the absence of any other viral protein. Fractionation experiments showed that Gag was present in cytoskeletal fraction containing long actin filaments and in a high-speed postcytoskeletal fraction with short actin filaments. We have also localized HIV-1 Gag to the lamellipodia of chemoattractant-stimulated cells. Significantly, the motility of Gag-expressing cells was enhanced in chemotaxis assays. In vitro mutagenesis experiments showed that HIV-1 Gag binds filamentous actin through the nucleocapsid domain (NC). An NC-green fluorescent protein fusion had the same cellular distribution as the complete protein, and its expression increased cell motility. These data suggest that interactions between HIV-1 Gag and actin in infected cells enhance cell motility. Ultimately this enhanced motility of infected cells could promote the dissemination of virus into the brain and other tissues.

3T3 Cells↗
PubMed2005

Differences in the length of gag proteins among different HIV type 1 subtypes.

The effect of HIV-1 subtype on Gag protein length was examined in 122 individuals infected with different HIV-1 clades. Except for the P1 protein, a wide variation in the Gag proteins length was noticed. P2 was significantly shorter in 68 non-B with respect to 54 subtype B viruses. Nearly 85% of subtype B gag sequences harbored P2 with 14 or more amino acid (aa) residues, while 75% of non-B subtypes had P2 with 13 or less aa (p < 0.0001). The P7 protein was one residue shorter in 64.2% of non-B specimens but only in 9.3% of subtype B isolates (p = 0.0001). Overall, the P6gag protein length was modified by the presence of insertions, deletions, and stop codons in 89 (73%) of the tested population, but was mainly dependent of changes in non- B compared to B viruses (97% vs. 42.6%, p < 0.0001). However, insertions at P6(gag) (from 1 to 9 aa) were significantly more frequent in B than in non-B viruses (33.3% vs. 4.4%; p = 0.00002). Overall, conserved Gag residues and aa motifs, regardless of the genetic subtype, were 68.7% in P1, 54% in P7, 33.3% in P2, and 25% in P6(gag) proteins. In summary, length variation in Gag proteins is extensive across different HIV-1 subtypes, and could influence protein structure and function. The effect of Gag variation on the viral cycle among different HIV-1 clades needs to be further investigated.

Amino Acid Motifs↗
PubMed2006

Enhancing efficacy of HIV gag DNA vaccine by local delivery of GM-CSF in murine and macaque models.

Controlled release of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific cytotoxic T lymphocyte (CTL) and T helper (Th) responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific antibody (Ab), Th, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and Th, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design.

Albumins↗