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PubMed1984

Exogenous glycosaminoglycans (GAG) are able to modulate avian skin differentiation (epithelial keratinization and feather formation).

Several reports have suggested that mesenchymal glycosaminoglycans (GAG) may be involved in the regulatory role of epithelial differentiation. Some researchers have pointed out that exogenous GAG affects extracellular GAG accumulation. We have therefore examined the effect of added GAG on two typical processes of avian skin differentiation: keratinization and feather formation. Glycosaminoglycans, either obtained from fibroblasts cultures (conditioned media) or purified commercially available GAG were administered to 5/6-day chick embryo back skin explants. Control cultures were supported with 199 synthetic medium, chick embryo extract or calf serum. Explants have been examined by histological and histochemical procedures. Skin explants maintained in vitro for 7 days exhibited an epithelial differentiation and a dermal histochemical reactivity which were related to the composition of the culture medium. In conditioned media from dermal fibroblasts, but not from heart or lung fibroblasts, explants always exhibited keratinization. In purified-GAG-containing media, keratinization was observed with condroitinsulphates and not with hyaluronic acid. Keratinization was always related to prevalent accumulation of hyaluronic acid in the underlying mesenchyme whereas feather formation was in relation to deposits of condroitinsulphates in dermis pulp. The above findings demonstrate that exogenous GAG is able to modulate avian skin differentiation and that this regulation is linked to an influence on the mesenchymal GAG pattern.

Animals↗
PubMed1990

Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV.

We constructed a recombinant vaccinia virus carrying the entire gag and pol genes of human immunodeficiency virus type 1 (HIV-1). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of 100-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pol proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pol gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production.

Animals↗
PubMed1991

Functional domains of HIV-1 gag-polyprotein expressed in baculovirus-infected cells.

Seven recombinants of AcNPV harboring various forms of complete or truncated gag gene from HIV-1 were constructed to determine which functional domains of the gag polyprotein are implicated in its self-assembly and cellular localization. The p6 carboxy-terminal portion of the p15 NCgag domain appeared to be dispensable for assembly, budding, and release of gag particles by insect cells. However, all the morphopoietic information was not entirely confined to the p9 NC domain, as N-myristylation could compensate for p15 NC deletion in gag assembly and the budding process. The two consensus karyophilic signals situated in the p17 MAgag domain were inefficient for targeting nonmyristylated forms of gag polyprotein to the nucleus when the p6 NC domain was deleted. In the presence of p6, or with a third, baculovirus-specific, karyophilic signal added at its N-terminus, gag particles relocated in the nucleus. These data suggested that p6 played a critical role in the conformation of gag polyprotein.

Amino Acid Sequence↗
PubMed2004

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles.

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.

APOBEC-3G Deaminase↗
PubMed2004

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.

Animals↗
PubMed2005

The virion-associated Gag-Pol is decreased in chimeric Moloney murine leukemia viruses in which the readthrough region is replaced by the frameshift region of the human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 translational frameshift event to synthesize the precursor of its enzymes, Gag-Pol, when ribosomes from the infected cells translate the full-length viral messenger RNA. Translation of the same RNA according to conventional translational rules produces Gag, the precursor of the structural proteins of the virus. The efficiency of the frameshift controls the ratio of Gag-Pol to Gag, which is critical for viral infectivity. The Moloney murine leukemia virus (MoMuLV) uses a different strategy, the programmed readthrough of a stop codon, to synthesize Gag-Pol. In this study, we investigated whether different forms of the HIV-1 frameshift region can functionally replace the readthrough signal in MoMuLV. Chimeric proviral DNAs were obtained by inserting into the MoMuLV genome the HIV-1 frameshift region encompassing the slippery sequence where the frameshift occurs, followed by the frameshift stimulatory signal. The inserted signal was either a simple stem-loop, previously considered as the stimulatory signal, or a longer bulged helix, now shown to be the complete stimulatory signal, or a mutated version of the complete signal with a three-nucleotide deletion. Although the three chimeric viruses can propagate essentially as the wild-type virus in NIH 3T3 cells, single-round infectivity assays revealed that the infectivity of the chimeric virions is about three to fivefold lower than that of the wild-type virions, depending upon the nature of the frameshift signal. It was also observed that the Gag-Pol to Gag ratio was decreased about two to threefold in chimeric virions. Comparison of the readthrough efficiency of MoMuLV to the HIV-1 frameshift efficiency, by monitoring the expression of a luciferase reporter in cultured cells, revealed that the frameshift efficiencies were only 30-60% of the readthrough efficiency. Altogether, these observations indicate that replacement of the readthrough region of MoMuLV with the frameshift region of HIV-1 results in virions that are replication competent, although less infectious than wild-type MoMuLV. This type of chimera could provide an interesting tool for in vivo studies of novel drugs targeted against the HIV-1 frameshift event.

Animals↗
PubMed2006

Downregulation of human immunodeficiency virus type 1 Gag expression by a gp41 cytoplasmic domain fusion protein.

The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, beta-galactosidase (beta-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli beta-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.

Cell Line↗
PubMed2000

Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

5' Untranslated Regions↗
PubMed1994

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Animals↗
PubMed1990

Incorporation of chimeric gag protein into retroviral particles.

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.

Amino Acid Sequence↗
PubMed1993

Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants.

We studied the effects of gag mutations on human immunodeficiency virus type 1 (HIV-1) assembly, processing, and infectivity by using a replication-defective HIV expression system. HIV mutants were screened for infectivity by transduction of a selectable marker and were examined for assembly by monitoring particle release from transfected cells. Gag protein processing and reverse transcriptase activities of mutant particles were also assayed. Surprisingly, most Gag protein mutants were assembled and processed. The two exceptions to this rule were a myristylation-minus mutant, and one gag matrix domain mutant which expressed proteins that were trapped intracellularly. Interestingly, a mutant with a 56-amino-acid deletion within the HIV gag capsid domain still could assemble and process virus particles, exhibited a wild-type retrovirus particle density, and had wild-type reverse transcriptase activity. Indeed, although most HIV-1 gag mutants were noninfectious or poorly infectious, they produced apparently normal particles which possessed significant reverse transcriptase activities. These results strongly support the notion that the HIV-1 Gag proteins are functionally involved in post-assembly, postprocessing stages of virus infectivity.

Amino Acid Sequence↗
PubMed1994

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Amino Acid Sequence↗
PubMedDate not supplied

[Interaction of gag polyprotein-precursors p55 and p48 with p160gag-pol during formation of virus-like particles of HIV-1 with recombinant strains of vaccinia virus].

The polypeptide composition of HIV-I virus-like particles produced by CV-I cells during mono- and coinfection with recombinant vaccinia virus (rVV) strains containing the whole (p55) and carboxyterminal truncated (p48) gag genes and gag-pol sequence is studied. In monoinfection both the gag-strains actively produced virus-like particles consisting of non-processed p55Gag and p48Gag polyprotein without p6 domain. In case of a coinfection of the cells with one of these strains and the rVV producing p160Gag-Pol polyprotein the virus-like particles consisted of p24 protein and a negligible amount of non-processed Gag precursors. The share of p24 protein increased in proportion to the duration of coinfection and decreased with a reduction of multiplicity of infection with rVV carrying p160Gag-Pol. Hence, the absence of p6 domain does not influence the processing of Gag proteins during virus-like particles assembly and budding. In contrast to the natural systems of HIV-I development, in the rVV expression system the p6Gag domain virtually does not contribute to reactions between Gag and Gag-Pol precursors and to the particles' morphogenesis.

Animals↗
PubMed1991

Humoral immune responses to gag and env proteins from human immunodeficiency virus type 1 in hemophiliac patients.

Solid-phase enzyme immunoassays using recombinant gag and env proteins were developed to study humoral immune responses to HIV infection in a cohort of 105 hemophiliac patients. Thirteen patients with ARC or AIDS and 92 asymptomatic patients were studied. A cross-sectional study showed a wide range of antibody responses to gag and env proteins; however, the differences between the ARC/AIDS and asymptomatic patients were statistically significant for both antigens (P less than .0004). In a longitudinal study, antibody levels in sera from 11 asymptomatic patients with gag antibody log units less than or equal to 1.5 were compared to levels in sera from 10 ARC/AIDS patients and 8 asymptomatic patients with gag antibody greater than 1.5. These patient groups were followed for comparable periods of time (67.1-71.7 mo). The asymptomatic patients with low gag antibody and the ARC/AIDS patients showed a similar pattern of antibody response to gag protein overtime. In hemophiliac patients with HIV-1 infection a low titer of antibody to gag protein is not invariably associated with clinical deterioration and is not a useful serologic marker of impending progression to AIDS.

AIDS-Related Complex↗
PubMed1995

Translation efficiency of the human T-cell leukemia virus (HTLV-2) gag gene modulates the frequency of ribosomal frameshifting.

The regulation of ribosomal frameshifting during translation of the polycistronic mRNA of human T-cell leukemia virus (HTLV) was studied in a cell-free system. Translation inhibitors such as cycloheximide and puromycin antibiotics were much more effective in blocking the synthesis of the frameshift polypeptide Gag-Pro than the synthesis of the Gag product. The preferential inhibition of the frameshift polypeptide Gag-Pro by the two antibiotics was not a reflection of the different sizes of the two gene products, but rather a consequence of the effect of the inhibitors on ribosomal translation efficiencies. To further analyze the effect of translation efficiencies on ribosomal frameshifting, we compared the translation of 5'-capped RNA to noncapped RNA. The translation of 5'-capped RNA was higher, as expected. Consequently, ribosomal frameshifting producing the Gag-Pro polypeptide was enhanced when compared to the translation of noncapped RNA. Taken together these results indicate that efficiencies of translation, in conjunction with the cis regulatory genetic elements at the frameshift sites, determine the ratio of the polypeptides Gag, Gag-Pro, and Gag-Pro-Pol produced in the HTLV-infected cell. Thus, physiological changes which affect the cellular translation machinery may alter the optimal ratio of these three polyprotein products needed for virus maturation.

Cell-Free System↗
PubMed1998

Gag protein from human immunodeficiency virus type 1 assembles in the absence of cyclophilin A.

Human immunodeficiency virus type 1 (HIV-1) replication requires coordinated activities of host and viral factors. We reported previously that interactions of the host factor cyclophilin A with HIV-1 Gag polyproteins affected Gag processing and maturation of virus particles (Streblow et al., 1998. Virology 245, 197-202). We now use in vitro translation and physical analysis of Gag structures to refine our understanding of how cyclophilin A affects HIV-1 replication. Gag assembled into oligomeric structures in vitro in the presence or absence of cyclophilin A, and proteins synthesized under the two conditions were equally susceptible to cleavage by exogenous HIV-1 protease. These and previous data show that Cyclophilin A is required at a step between Gag assembly and Gag processing/virion morphogenesis. Cyclophilin A may be required for Gag conformational changes subsequent to assembly, that are required for efficient dimerization and activation of the viral protease.

Animals↗
PubMed2002

Interaction of HIV-1 gag and membranes in a cell-free system.

A coupled transcription-translation (TNT) reticulocyte lysate system was used to examine posttranslational alterations in HIV-1 Gag upon addition of Jurkat T cell membranes. Incubation of the Gag precursor protein, Pr55gag, with membranes resulted in a time-dependent alteration in Gag resulting in partial resistance to trypsin treatment. Treatment of membranes and TNT extract with apyrase or pretreatment of membranes with trypsin prevented this posttranslational alteration of Gag. In contrast, this activity was not disrupted by pretreatment of membranes with Triton X-100 at 4 degrees C, under conditions which do not solubilize raft-associated proteins. Flotation studies revealed that acquisition of trypsin-resistance was accompanied by Gag binding to membranes. The myristylation signal and nucleocapsid domain were found to mediate Gag binding to membranes. The posttranslational alteration of Gag accompanying membrane interaction may represent a conformational change, oligomerization, and/or association with or envelopment by membranes. These findings provide new clues to the stepwise process of HIV-1 assembly.

Adenosine Triphosphate↗
PubMed2003

Time course of Gag protein assembly in HIV-1-infected cells: a study by immunoelectron microscopy.

Recent biochemical studies have identified high molecular complexes of the HIV Gag precursor in the cytosol of infected cells. Using immunoelectron microscopy we studied the time course of the synthesis and assembly of a HIV Gag precursor protein (pr55gag) in Sf9 cells infected with recombinant baculovirus expressing the HIV gag gene. We also immunolabeled for pr55gag human T4 cells acutely or chronically infected with HIV-1. In Sf9 cells, the time course study showed that the first Gag protein appeared in the cytoplasm at 28-30 h p.i. and that budding started 6-8 h later. Colloidal gold particles, used to visualize the Gag protein, were first scattered randomly throughout the cytoplasm, but soon clusters representing 100 to 1000 copies of pr55gag were also observed. By contrast, in cells with budding or released virus-like particles the cytoplasm was virtually free of gold particles while the released virus-like particles were heavily labeled. Statistical analysis showed that between 80 and 90% of the gold particles in the cytoplasm were seen as singles, as doublets, or in small groups of up to five particles probably representing small oligomers. Clusters of gold particles were also observed in acutely infected lymphocytes as well as in multinuclear cells of chronically infected cultures of T4 cells. In a few cases small aggregates of gold particles were found in the nuclei of T4 lymphocytes. These observations suggest that the Gag polyprotein forms small oligomers in the cytoplasm of expressing cells but that assembly into multimeric complexes takes place predominantly at the plasma membrane. Large accumulations of Gag protein in the cytoplasm may represent misfolded molecules destined for degradation.

Animals↗