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At least 19 recordsLinked to original sources

PubMed1990

The simian immunodeficiency virus (SIV) rev gene regulates env expression.

The rev gene product is a trans-acting nuclear regulatory protein that is essential for AIDS virus replication. To define the effect of the SIV rev gene on its gp160 expression, two SV40 constructs have been made: pBC17 is a rev+env+ construct, and pBD21 is a rev-env+ construct. After transfecting the constructs into COS-1 cells, RNA, protein, syncytium formation, and monoclonal antibody blocking assays were performed. The results indicated that the SIV rev gene positively influenced the level of full-length env mRNA and was required for expression of SIV gp160.

Antibodies, Monoclonal↗

PubMed1992

HIV-1 gene rev responsible for accumulated heterodisperse RNAs seen in infected T-cells.

To analyze which gene of HIV-1 was responsible for the high accumulation of "heterodisperse RNAs" in virally infected T-cells, Northern blot analysis was performed. Total RNA from inducible cell lines expressing the envelope protein of HIV-1, or the regulatory gene rev, were blotted and hybridized with an Alu probe that detects heterodisperse RNAs. Results show that the rev gene is responsible for the accumulation of cellular heterodisperse RNAs seen earlier in HIV-1-infected T-cells.

Blotting, Northern↗

PubMed1991

Compatibility of rev gene activity in the four groups of primate lentiviruses.

The compatibility of rev genes derived from various primate immunodeficiency viruses of all distinct subgroups identified was assessed in three experimental systems: complementation experiments between proviral rev and gag mutants, evaluation of the ability of the rev gene products to activate proviral reporters, and examination of the capacity of various viruses to augment marker gene expression in the infected reporter cell lines. In all systems, human immunodeficiency virus type 1 (HIV-1) rev was not substantially substituted or was extremely poorly substituted by the rev of the other viruses. The rev of simian immunodeficiency virus (SIV) from a mandrill could be exchanged by HIV-1 rev. In contrast, the rev gene products of all viruses efficiently activate HIV-2 and SIV from an African green monkey.

Base Sequence↗

PubMed1990

Complementation of the rev gene mutation among human and simian lentiviruses.

The functional exchangeability of the rev gene was assessed in transient transfection experiments by using in vitro-constructed rev and gag mutants of the following three primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus from the African green monkey (SIV AGM). Cotransfection into SW480 cells of the rev and gag mutants derived from the DNA of each infectious virus resulted in the generation of progeny particles as determined by reverse transcriptase assay. rev gene mutants of HIV-2 and SIV AGM were also complemented by all gag mutants derived from the three viruses. In contrast, no evidence of complementation was obtained following cotransfection of the HIV-1 rev mutant and the gag mutant of HIV-2 or SIV AGM.

Amino Acid Sequence↗

PubMed1990

Subcellular localization of rev-gene product in visna virus-infected cells.

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.

Animals↗

PubMed1990

High level expression of the envelope glycoproteins of the human immunodeficiency virus type I in presence of rev gene using helper-independent adenovirus type 7 recombinants.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Adenoviridae↗

PubMed1989

Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method.

Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modified the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-1JR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-1JR-CSF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.

Base Sequence↗

PubMed1994

The origin of human immunodeficiency virus type-1 rev gene. An evolutionary hypothesis.

The Rev protein of human immunodeficiency virus type-1 is an RNA-binding posttranscriptional transregulator encoded by an accessory gene that is distinct from retroviral oncogenes and whose origin is unclear. We hypothesize that the rev gene was generated by duplication of a viral RNA segment having a secondary-structure that evolved into the Rev-responsive element (RRE). This hypothesis is based on the following findings. First, accumulated data on functional mapping of Rev, Tat, and the transmembrane protein of Env suggested that the major coding exon of rev should have been inserted into the transmembrane region of env during the course of its evolution. Experiments with equine infectious anemia virus, another complex retrovirus, also indicate that a large portion of rev is located within the dispensable transmembrane region of env. Second, base usage analysis suggests the same origin for rev and RRE. Our hypothesis may provide a new insight into the evolutionary aspect of RNA-binding transactivators.

Base Sequence↗

PubMed1989

Low antigenicity of HIV-1 rev: rev-specific antibody response of limited value as correlate of rev gene expression and disease progression.

An enzyme immunoassay based on an E. coli-produced HIV-1 rev gene product was used to detect rev-specific antibodies in longitudinally collected serum samples from 196 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. In 61% of men no rev-specific antibodies were detected at all, 30% had persistently detectable rev-specific antibodies, and in 9% rev-specific antibodies were only transiently or intermittently detected. When a persistent rev-specific antibody response occurred in subjects who seroconverted to structural proteins, it was always, with one exception, found within 12 months of seroconversion. The rev-specific antibodies were also studied in a transectional sample of sera from the men who remained symptom-free and from those who developed AIDS-related conditions or AIDS, as well as in sera from 31 other men with AIDS-related conditions and in sera from 6 of these men at the time they developed AIDS. The rev-specific antibodies were found in 34% of symptom-free men, in 28% of patients with AIDS-related conditions, and in 16% of patients with AIDS. The low incidence of rev-specific antibodies early after infection may be due to low antigenicity of rev. The lower prevalence of rev-specific antibodies in sera from patients with AIDS, compared with patients with AIDS-related conditions and symptom-free HIV-1-infected individuals, may be explained by a progressive HIV-1-induced immunodeficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

PubMed1990

Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization.

The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.

Amino Acid Sequence↗

PubMed1994

The rev gene of visna virus is required for productive infection.

To assess the role of the animal lentivirus rev regulatory gene product in the life cycle of visna virus, we introduced mutations into the functional fourth exon of the rev gene of visna virus. Cultured cells transfected with a full-length clone of visna DNA produce infectious virus but visna DNA with mutations in rev does not. The documented requirement for a functional rev gene in productive infections establishes the essential role of this gene in the replication of an animal lentivirus.

Amino Acid Sequence↗

PubMed1993

Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1.

Mice developed massive splenomegaly and polyclonal hypergammaglobulinemia within 2 days after intravenous injection of a phosphorothioate oligomer that is antisense to a portion of the rev region of the HIV-1 genome. Histologic examination of spleens from injected animals showed marked expansion of a uniform-appearing population of small lymphocytes and many mitoses. Spleen mononuclear cells (SMNCs) from injected animals showed approximately a 10-fold-increased uptake of [3H]thymidine and production of IgM and IgG. Flow cytometry analysis indicated that the responding cells were predominantly B-lymphocytes. The anti-rev oligomer also was mitogenic in vitro and stimulated immunoglobulin production by normal mouse SMNCs and human peripheral blood mononuclear cells. Similar immunologic effects were observed with an anti-rev 21-mer phosphorothioate, truncated at the 3' end, but not with a 20-mer human p53 antisense phosphorothioate or a 28-mer anti-rev phosphodiester. These observations are consistent with the possibility that DNA sequences homologous to the rev gene participate in the regulation of mammalian lymphocyte activation, proliferation and maturation.

Animals↗

PubMed1991

Identification of feline immunodeficiency virus rev gene activity.

We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.

Animals↗

PubMed1988

Phosphorylation of the rev gene product of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

B-cell proliferation and differentiation in common variable immunodeficiency patients produced by an antisense oligomer to the rev gene of HIV-1.

The immunostimulatory activity of a phosphorothioate oligodeoxynucleotide (27 mer) that is antisense to the rev gene of HIV-1 was studied on normal human lymphocytes and on cells from patients with common variable immunodeficiency (CVI). For peripheral blood mononuclear cells from nine normal individuals, the proliferation index (16.8 +/- 12.5) after anti-rev oligomer exposure was proportional to the percentage of peripheral B-cells (r = 0.76, P = 0.02). In five experiments, enriched B- or T-cell populations had proliferation indices of 47.2 +/- 32.9 and 2.4 +/- 1.9, respectively. The addition of T-cells to anti-rev oligomer treated B-cells had no effect (proliferation index = 47.5 +/- 38.1). After anti-rev oligomer stimulation, autoradiography, and counterstaining for B- and T-cell markers, all detectable [3H]thymidine uptake was by CD19-positive cells. Eight of the 14 CVI patients had a proliferation index and secreted levels of IgM and IgG comparable to cells from normal individuals. In contrast to normal cells, the direct correlation between proliferation of peripheral blood mononuclear cells and the percentage of peripheral B-cells was weak in samples from 13 CVI patients (r = 0.4, P = 0.2). These findings indicate that peripheral blood B-cells from about half of CVI patients proliferate and produce immunoglobulin after exposure to anti-rev oligomer. These data demonstrate that under the appropriate circumstances, B-cells of some CVI patients can proliferate and differentiate normally.

B-Lymphocytes↗

PubMed1994

Effect of mutations in rev gene of SIVmac on virus replication.

The functional activity of SIVmac251 Rev was altered by introducing amino acid changes inside and chain termination mutations after the Rev response element-binding region (RBR) of the protein. The effects of specific mutations were evaluated by transfecting proviral DNAs into the HeLa cell line and into HeLa cells constitutively expressing either HIV-1 Rev or HTLV-1 Rex proteins. Cell-free supernatants from these transient expression assays were further characterized by infecting CD4-positive lymphoid cell lines H9 and MT-4, the latter abortively infected with HTLV-1, and human peripheral blood mononuclear cells. These results together with the data from cotransfection experiments show that SIV can be attenuated up to 95% by introducing changes into the arginine-rich domain, RBR, of Rev. These recessive mutations were efficiently complemented in trans by HIV-1 Rev, SIV Rev, and HTLV-I Rex proteins. In contrast, the mutants of Rev protein that had a chain termination after RBR were trans-dominant negative and could not be trans-complemented with any of these three regulatory proteins. When additional mutations were inserted into the RBR of these trans-dominantly negative Rev proteins, complementation was obtained again.

Amino Acid Sequence↗

PubMed1990

Coexpression of biologically active simian immunodeficiency virus (SIV) Rev and Env in an SV40 system: the SIV rev gene regulates env expression.

The coexpression of biologically active simian immunodeficiency virus (SIV) Rev and Env gene products was obtained in COS-1 cells from a single SIV subgenomic segment (which contains both exons of rev and the entire env gene) cloned into a SV40-directed vector. The SIVsm Rev trans-activated the expression of the full-length env mRNA and was required for the production of envelope glycoproteins. Furthermore, the alignment of the structural conservation of the Rev functional domains among all HIV and SIV was analyzed.

Amino Acid Sequence↗

PubMed1995

Persistence of attenuated rev genes in a human immunodeficiency virus type 1-infected asymptomatic individual.

With the goal of examining the functional diversity of human immunodeficiency virus type 1 (HIV-1) env genes within the peripheral blood mononuclear cells of an asymptomatic individual, we substituted four complete env genes into the replication-competent NL4-3 provirus. Despite encoding full-length open reading frames for gp120 and gp41 and the second coding exon of tat and rev, each chimera was replication defective. Site-directed mutagenesis of codon 78 in the Rev activation domain (from a hitherto unique Ile to the subtype B consensus Leu) partially restored infectivity for two of three chimeras tested. Similarly, mutagenesis of rev codon 78 of NL4-3 from Leu to Ile partially attenuated this virus. Ile-78 was found in all 13 clones examined from samples taken from this asymptomatic subject 4.5 years after infection, including 9 from peripheral blood mononuclear cells and 4 from a virus isolate, as well as 4 additional clones each from peripheral blood mononuclear cells sampled 37 and 51 months later. We next examined conservation of the Rev activation domain within and among long-term survivors (LTS) and patients with AIDS, as well as T-cell-line-adapted strains of HIV-1. Putative attenuating mutations were found in a minority of sequences from all five LTS and two of four patients with AIDS. Of the 11 T-cell-line-adapted viruses examined, none had these changes. Among and within LTS virus population had marginally higher levels of diversity in Rev than in Env; patients with AIDS had similar levels of diversity in the two reading frames; and T-cell-line-adapted viruses had higher levels of diversity in Env. These results are consistent with the hypothesis that asymptomatic individuals harbor attenuated variants of HIV-1 which correlate with and contribute to their lack of disease progression.

Acquired Immunodeficiency Syndrome↗