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At least 19 recordsLinked to original sources
PubMed1984

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.

Amino Acid Sequence↗
PubMed1983

A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens.

From molecularly cloned DNAs of Fujinami sarcoma virus (FSV) and the Schmidt-Ruppin-A strain of Rous sarcoma virus (SRA), viral DNA was constructed in which fps-specific sequences encoded in FSV replaced the src gene of SRA. A 3' fragment of FSV DNA, from an ATG methionine coding sequence 148 base pairs downstream from the gag-fps junction through the long terminal repeat, was joined to cloned SRA DNA at the translation start site for the src gene. The resultant DNA clone contained the splice acceptor site for src mRNA processing in SRA, but contained no src coding sequences from SRA nor any gag sequences from FSV. All genes for the replication of SRA were retained. Transfection of this cloned viral DNA genome into chicken embryo fibroblasts induced morphological transformation of the cells in culture. However, the morphology of the transformed cells was distinct from that observed in cells infected with wild-type FSV. The transformed cells produced a nondefective transforming virus called F36 which contained a hybrid FSV-SRA long terminal repeat. F36-infected cells produced a protein with the expected molecular weight of 91,000, which had an associated protein kinase activity and was immunoprecipitated by antibodies raised against fps gene determinants but not by antibodies raised against gag or src proteins. Injection of F36 virus into 8-day-old chicks produced tumors at the site of inoculation, detectable within 7 days. These results demonstrated that the gag portion of the gag-fps fusion protein of FSV is not required for transformation or tumorigenesis.

Animals↗
PubMed1994

Site-directed mutagenesis of the P2 region of the Rous sarcoma virus gag gene: effects on Gag polyprotein processing.

The Pr76Gag and Pr180Gag-Pol polyprotein precursors of Rous sarcoma virus contain a 22-amino-acid spacer peptide, called p2, located between the amino acid sequences of the mature Gag proteins MA and p10. This spacer peptide is present in stoichiometric amounts in the virion, albeit cleaved into two parts, but its function is unknown. The primary sequence of this peptide includes a region that is highly conserved among retroviruses, consisting of four prolines followed by tyrosine. We have investigated the role of p2, particularly the polyproline motif, in the virus life cycle by site-directed mutagenesis. Mutations in this region result in the intracellular accumulation of a truncated Gag precursor, due either to a block in the intracellular processing of the precursor or to the premature activation of the viral protease. Since in cells infected by Rous Sarcoma Virus there is no significant intracellular processing of the Gag polyprotein precursor, our data suggest that the p2 domain plays a role in controlling the activation of the protease. These mutations also result in a reduction in virus particle release, probably as a direct consequence of the aberrant precursor processing since a construct in with both p2 and the protease active site were mutated did not exhibit aberrant processing of the Gag polyproteins and formed particles with an efficiency similar to that of the wild type. This indicates that it is the viral protease that is responsible for the aberrant processing and suggests that the p2 region is not required for assembly. Although the virus genomic RNA packaged into virions produced by the p2 mutants is more susceptible to degradation, it appears that the p2 domain does not have a direct role in RNA packaging and protection.

Amino Acid Sequence↗
PubMed1991

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

Animals↗
PubMed1994

A p12 gag gene homologue is present in the mouse genome.

A replication-defective virus (BM5d) of approximately 4.9 kb, is responsible for a retrovirus induced immunodeficiency syndrome in mice (MAIDS) that shares many features with AIDS. BM5d is characterized by deletions in env and pol genes, furthermore its gag gene differs markedly from gag of other BM5 ecotropic viruses, particularly in its p12 sequence. The p12 region of the gag gene has been shown to account for the pathogenicity of the BM5d retrovirus. During our studies of BM5d integration in mice we found that p12-like sequences are present in the mouse genome of uninfected healthy C57BL/6 mice. Cloning and sequencing of this p12 gag homologue has revealed a high (63% to 89%) amino acid derived sequence identity with other retroviruses and shown that the major differences among p12 of pathogenic viral strains compared to non-pathogenic ones consist of a four amino acids deletion and a high abundance of proline and basic amino acids in their p12 region.

Amino Acid Sequence↗
PubMed1994

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Animals↗
PubMed2003

Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting.

Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of gag remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the gag region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus gag, some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral gag gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.

Amino Acid Sequence↗
PubMed1985

Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins.

Five gag-gene-encoded structural proteins, designated p12, pp18, pp20, p30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroform-methanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NH2- and COOH-terminal sequence analysis p12, pp18, p30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NH2-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phospho-proteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gag-precursor polyprotein, Pr60gag in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60gag, viz., NH2-p12-pp18-p30-p10-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5'- and 3'-gag regions (i.e., p12 and p10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques.

Amino Acid Sequence↗
PubMed1990

Rous sarcoma virus expression in Saccharomyces cerevisiae: processing and membrane targeting of the gag gene product.

In avian cells, the product of the gag gene of Rous sarcoma virus, Pr76gag, has been shown to be targeted to the plasma membrane, to form virus particles, and then to be processed into mature viral gag proteins. To explore how these phenomena may be dependent upon cellular (host) factors, we expressed the Rous sarcoma virus gag gene in a lower eucaryote, Saccharomyces cerevisiae, and studied the behavior of the gag gene product. We show here that Pr76gag is processed in yeast cells and that this processing is specific, since it is abolished in a mutant in which the active site of the gag protease has been destroyed. In this mutant, the uncleaved precursor is found associated with the yeast plasma membrane, yet no virus particles were detected in cells or in the culture medium. From our results, we can speculate either that in yeast cells, a host protease initiates Pr76gag processing in the cytosol or that in avian cells, an inhibitor prevents the processing until the viral particle is formed.

Avian Sarcoma Viruses↗
PubMed2005

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene.

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. CONCLUSION: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

AIDS Vaccines↗
PubMed2000

[Cloning and expression of full-length subtype E and subtype B gag gene from uncultured PBMCs of HIV-1 infected individuals in China].

OBJECTIVE: To clone and express subtype E and subtype B gag gene of the prevalent HIV-1 strain in China. METHODS: One HIV-1 positive sample, whose subtype had been determined as sub-type E and one HIV-1 positive sample, whose subtype had been determined as subtype B by sequence analysis of partial env gene were chosen to be amplified by nested PCR. We got two full-length fragments of gag gene and incorporated them first into plin8Pr55 and then into pFastBacl. RESULTS: Sequence analysis showed the cloned gag genes have the complete open-reading frames and have no major deletion and insertion. Using the Bac-toBac system, we obtained the recombinant baculoviruses containing gag genes. Western-blot analysis showed that insect cells infected with gag recombinant baculoviruses expressed HIV-1 gag antigen. Thin section electron microscopy showed that virus-like particles (VLPs) formed in insect cells was infected with gag. CONCLUSIONS: The cloning and expression of gag genes of the prevalent HIV-1 stains in China revealed that the cloned genes have intact structure and function and this paves the way for the development of HIV-1 vaccines targeted at the epidemic of HIV-1 in China, also for the study of biological functions of gag gene and development of diagnostic kits of HIV-1.

Animals↗
PubMed1978

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↗
PubMed1984

Three independent polyproteins coded by the gag gene of type-D retrovirus in a continuous human cell line.

The processing of gag-gene-coded polyproteins of type D retrovirus (HEp-2 V) in chronically infected continuous human larynx carcinoma cell line HEp-2 (HeLa-like) was investigated by means of the pulse-chase modification of the radioimmunoprecipitation test. Three independent polyproteins coded by the gag gene of HEp-2 V were revealed in the immunoprecipitates: Pr 78gag, a direct precursor of the virus internal structural polypeptides, described in a previous report (1); Pr 180gag + pol, a probable reverse transcriptase precursor; and gPr 78gag, a glycosylated polyprotein of unknown function. Two unstable intermediate polyproteins derived from Pr 78gag cleavage were detected in the presence of serine protease inhibitors. These polypeptides, having molecular weights of 37 K and 33 K, are Pr 37gag and Pr 33gag respectively. A probable scheme of gag-gene-coded polyproteins processing is suggested, and speculations on the gag-gene-coded glycosylated polyproteins of retroviruses as growth factor receptors are also presented.

Cell Line↗
PubMed1995

Expression of feline immunodeficiency virus (FIV) gag gene in vaccinia virus vector.

The selected part of gag gene (nucleotide sequences at position 618 to 1724) coding for P24 and P15 proteins of feline immunodeficiency virus (FIV) was expressed in vaccinia virus vector under control of 7.5 kDa vaccinia virus transtriptional signal (p 7.5 vaccinia virus early/late promoter). The vaccinia virus recombinant (denoted VV-G(1)) was generated by homologous recombination following CV-1 cells transfection with a newly prepared vaccinia virus insertion vector -- pWKr75.

Animals↗
PubMed1996

Intrapatient sequence variation of the gag gene of human immunodeficiency virus type 1 plasma virions.

Because certain regions of the gag gene, such as p24, are highly conserved among human immunodeficiency virus (HIV) isolates, many therapeutic strategies have been directed at gag gene targets. Although intrapatient variation of segments of gag have been determined, little is known about the variability of the full-length gag gene for HIV isolated from a single individual. To evaluate intrapatient full-length gag variability, we derived the nucleotide sequences of at least 10 cDNA gag clones of virion RNA isolated from plasma for each of four asymptomatic HIV type 1-infected patients with relatively high CD4+ T-cell counts (300 to 450 cells per mm3). Mean values of intrapatient gag nucleotide variation obtained by pairwise comparisons ranged from 0.55 to 2.86%. For three subjects, this value was equivalent to that reported for intrapatient full-length env variation. The greatest range of intrapatient mean nucleotide variation for individual protein-coding regions was observed for p7. We did not detect any G-to-A hypermutation, as A-to-G and G-to-A transitions occurred at similar frequencies, accounting for 29 and 25%, respectively, of the changes. Mean variation values and phylogenetic analysis suggested that the extent of nucleotide variation correlated with the length of viral infection. Furthermore, no distinct subpopulations of quasispecies were detectable within an individual. The predicted amino acid sequences indicated that there were no regions within a gag protein that were comprised of clustered changes.

Amino Acid Sequence↗
PubMed1992

Immunological characterization of the gag gene products of bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.

Amino Acid Sequence↗
PubMed1992

Expression of feline immunodeficiency virus gag gene in Escherichia coli.

The gag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed in Escherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIV gag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.

Cloning, Molecular↗
PubMed1997

CM-gag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene.

CM-gag elements constitute an homogeneous family of sequences that are reiterated in the genome of Culex pipiens strains from different continents. Apparently complete 1.75 kb CM-gag copies are flanked by target-site duplications and have a polyadenylation signal near their 3' end. They potentially contain a unique gene encoding a putative protein that displays homologies with nucleic acid binding proteins and the gag polypeptide of retroviruses and retrotransposons, but that does not encode a reverse transcriptase. CM-gag elements are similar in their genetic organization to the telomeric transposable sequences Het-A from Drosophila melanogaster, but Southern-hybridization patterns indicate that the former are more probably dispersed in various areas of the mosquito genome. The homogeneity of CM-gag copies that are distributed worldwide suggests that they have most probably been amplified recently. Furthermore, selective constraints against amino acid changes have been acting on these sequences, suggesting that they need to encode the gag-like protein to be incorporated into the chromosomes.

Amino Acid Sequence↗