The human immunodeficiency virus type 1 Vpr transactivator: cooperation with promoter-bound activator domains and binding to TFIIB.

Since the first report documenting that HIV-1 Vpr was involved in the stimulation of transactivation of several unrelated promoters, little additional information has been reported. By using transient transfection experiments, we confirmed and extended these previously reported data. Further in vivo experiments showed that Vpr can co-operatively stimulate transactivation activity of a minimal promoter containing one GAL4 DNA-binding site, when it is co-expressed with different heterologous activator domains fused to GAL4 DNA-binding domain. Thus, Vpr could transactivate in concert with an activator domain, but has no effect on the transactivation of a minimal promoter in the absence of activator protein. To investigate whether Vpr can interact with components of the basal transcriptional machinery, in vitro protein-protein binding assays were performed using either translated, radiolabeled Vpr or TFIIB proteins and glutathione S-transferase Vpr or TFIIB chimeric proteins. We demonstrated that the portion of Vpr ranging from amino acids 15 to 77 interacts specifically with the basal transcription factor TFIIB. Also, our data indicated that the N-terminal domain of TFIIB is required for the interaction.

Production of HIV-1 by resting memory T lymphocytes.

BACKGROUND: The persistence of HIV-1 within resting memory CD4 T cells constitutes a major obstacle in the control of HIV-1 infection. OBJECTIVE: To examine the expression of HIV-1 in resting memory CD4 T cells, using an in-vitro model. DESIGN AND METHODS: Phytohaemagglutinin-activated peripheral blood mononuclear cells were challenged with T cell-tropic and macrophage-tropic HIV-1 clones, and with a replication-incompetent and non-cytotoxic HIV-1-derived vector (HDV) pseudotyped by the vesicular stomatitis virus glycoprotein G. To obtain resting memory CD4 T cells containing HIV-1 provirus, residual CD25(+), CD69(+) and HLA-DR(+) cells were immunodepleted after a 3 week cultivation period. RESULTS: In spite of the resting phenotype, the majority of provirus-harbouring T cells expressed HIV-1 genomes and produced infectious virus into cell-free supernatant. The expression of HDV dropped by only 30% during the return of activated HDV-challenged cells into the quiescent phase. Although resting memory T cells generated in vitro expressed HIV-1 and HDV genome when infected during the course of the preceding T cell activation, they were resistant to HIV-1 and HDV challenge de novo. The infected culture of resting memory T cells showed a higher resistance to the cytotoxic effects of HIV-1 in comparison with the same cultures after reactivation by phytohaemagglutinin. CONCLUSION: The majority of resting memory T cells infected during the course of a preceding cell activation produces virus persistently, without establishing a true HIV-1 latency. The described system could be used as a model for testing new drugs able to control residual HIV-1 replication in resting memory T cells.

The HIV-1 Vpr co-activator induces a conformational change in TFIIB.

Vpr is a HIV-1 virion-associated protein which plays a role in viral replication and in transcription and cell proliferation. We have previously reported that Vpr stimulates transcription of genes lacking a common DNA target sequence likely through its ability to interact with TFIIB. However, the molecular mechanism of the Vpr-mediated transcription remains to be precisely defined. In this in vitro study, we show that the binding site of Vpr in TFIIB overlaps the domain of TFIIB which is engaged in the intramolecular bridge between the N- and C-terminus of TFIIB, highly suggesting that binding of Vpr may induce a change in the conformation of TFIIB. Indeed, with a partial proteolysis assay using V8 protease, we demonstrate that Vpr has the ability to change the conformation of TFIIB. We investigated in this partial proteolysis assay a series of Vpr-mutated proteins previously defined for their transactivation properties. Our data show a correlation between the ability of Vpr-mutated proteins to stimulate transcription and their ability to induce a conformational change in TFIIB, indicating a functional relevance of the Vpr-TFIIB interaction.

Neurotoxicity of peptide analogues of the transactivating protein tat from Maedi-Visna virus and human immunodeficiency virus.

Infection by lentiviruses such as human immunodeficiency virus, Maedi-Visna virus and Caprine Arthritis Encephalitis Virus, is associated with a variety of neurological syndromes, but the mechanism by which the damage occurs to the nervous system is not known. The viruses do not infect neurons and so the neurotoxic actions must be mediated indirectly. Here we applied synthetic peptide analogues derived from basic regions of Maedi-Visna virus and human immunodeficiency virus transactivating protein, tat, to rat brain in vivo and found them to be potent neurotoxins. The toxicity of the Maedi-Visna virus peptide was demonstrated to be reduced by blockade of nitric oxide synthase and of N-methyl-D-aspartate channel opening. These experiments suggest that peptides derived from lentiviral tat may share a common neurotoxic action.

Genetic variation and host markers in the src gene of recovered avian sarcoma viruses.

The src genes of three recovered avian sarcoma viruses were compared by RNase T1 oligonucleotide fingerprinting and tryptic peptide analysis. In all three recovered avian sarcoma viruses the oligonucleotide composition of src was different and also distinct from that of the parental Schmidt-Ruppin strain of Rous sarcoma virus. This evidence for genetic variation src was strengthened by two dimensional peptide maps of the src gene products pp60src, translated in a reticulocyte lysate system in vitro. Numerous differences between the peptide patterns of the pp60src proteins produced by the parental and the recovered viruses were detected. No two src proteins were identical, while the tryptic peptide maps of the internal gag proteins synthesized by these viruses were indistinguishable. The src proteins of recovered avian sarcoma viruses also contained peptides that were absent from the src protein of parental Schmidt-Ruppin D virus but were found in the endogenous src protein of normal cells. We conclude that there is considerable genetic variation in the src gene of recovered avian sarcoma viruses and that these recovered src genes contain host cell-derived markers.

[Experimental studies on maedi-visna]. / Recherches expérimentales sur le maedi-visna.

In order to study pathogenicity of sheep lentiviruses, to obtain monospecific sera and to perfect ELISA, 3 experiments with different strains were carried out for 4 yr. In expt 1, one clone only of a French maedi-visna strain (564-79) elicits a clear seroconversion in inoculated sheep. In expt 2, K1514 is more immunogenic than K796 and PPV: intratracheal route seems more efficient than intracerebral route. Sheep infected by ts mutants (expt 3) are early positive as wild strain K796. Nevertheless, the level of positivity is less important than for the parental strain, suggesting that the defect of the ts mutants is not limiting their replication in vivo. An important result is the lack of clinical signs and anatomical and histopathological lesions, in spite of frequent isolations of virus from buffy coat cells. These results suggest that: different enhancing factors have to be taken in account in the apparition of clinical signs; all the clones are not infectious; viral infection might be effective with several types of virions.

[An ELISA test for detection of maedi-visna antibodies. Comparative study with gel immunodiffusion and complement-fixation test]. / Une technique ELISA pour la détection des anticorps anti-virus maedi-visna. Etude comparative avec l'immunodiffusion en gelose et la fixation du complement.

An indirect microELISA test was performed for detection of maedi-visna antibodies in ovine and caprine species. The antigen consisted in viral particles, highly purified by successive ultracentrifugations. By comparative testing of 934 sera in ELISA and gel immunodiffusion, we found a good correlation between these two tests, and moreover, ELISA revealed another 11.3% of positive samples. The precocity of this ELISA was shown by experimental infection of sheep with different strains of maedi-visna: positive sera were detected 7 weeks post-infection, instead 4-5 months with gel immunodiffusion. The complement fixation test was compared with gel immunodiffusion and was found the less sensitive. This ELISA test appeared to be satisfactory, and may be used for early diagnosis of maedi-visna infection.

Isolation and identification of a South African lentivirus from jaagsiekte lungs.

In the course of attempts to grow the jaagsiekte retrovirus in cell culture, a typical lentivirus was isolated for the first time in South Africa from adenomatous lungs. Morphologically the virus could not be distinguished from other lentiviruses, but serologically it was shown to be more closely related to visna virus than to caprine arthritis-encephalitis virus. However, a preliminary restriction enzyme analysis of the linear proviral DNA of this new lentivirus (SA-DMVV) revealed that it is significantly district from visna virus and CAEV and therefore may represent a third type of lentivirus. Antibodies to the virus were demonstrated in a number of sheep in various parts of the country, but a direct link to a disease condition was not found. Attempts to produce lung lesions by intratracheal injection of the virus have been unsuccessful to date but a transient arthritis was produced by intraarticular inoculation. Viral replication seems to be enhanced in jaagsiekte lungs.

Properties of visna virus particles harvested at short time intervals: RNA content, infectivity, and ultrastructure.

The major RNA component of Visna virus harvested at short intervals of time (5 min) is not the 60 to 70S RNA but a molecule of higher electrophoretic mobility. This RNA has been isolated and characterized. Its sedimentation coefficient is identical to that of 30 to 40S RNA subunits obtained by heat denaturation of the 60 to 70S RNA. In 1.8% acrylamide gels without agarose the electrophoretic mobility of 30 to 40S RNA subunits present in rapidly harvested virus is slightly lower than that of the subunits obtained by denaturation of the 60 to 70S RNA; after heat denaturation the mobilities are identical. These free RNA subunits present in early virus particles assemble into a 60 to 70S RNA complex as shown by following the RNA content of early virus incubated at 37 C for various lengths of time. The rate of this maturation process is slow. There is no difference between the infectivity of immature and mature virus particles. Both particles have a dense core when examined in sections of virus pellets.

Partial enzyme digestion studies on Escherichia coli, Pseudomonas, Chlorella, Drosophila, HeLa and yeast 5S RNAs support a general class of 5S RNA models.

Fox and Woese (1975a) have shown that a model of 5S RNA secondary structure similar to the one originally derived for Chlorella 5S RNA can be generalized with relatively minor variations to all sequenced 5S RNA molecules, i.e. that corresponding base paired regions can be formed at approximately the same positions. We present experimental data in favour of this hypothesis and show that the points at which ribonucleases T1, T2 and pancreatic ribonuclease cleave six different 5S RNA molecules under 'mild' conditions (high ionic strength, low temperature, low RNAase concentration) nearly always fall in the proposed single-stranded regions. We conclude that this model is a good approximation to the conformation of 5S RNA in solution.

The visna transcriptional activator Tat: effects on the viral LTR and on cellular genes.

U937 promonocytic cells, either treated or untreated with phorbol-esters, were used for transient expression assays. We analyzed a series of visna LTR plasmids containing either the AP-1 or the AP-4 or both target responsive sequences for visna Tat transactivation. A 5' deletion mutant of the LTR containing a truncated AP-4 target sequence lost the Tat-mediated transactivation, while phorbol ester-mediated transactivation was not affected. Furthermore, the absence of this AP-4 sequence dramatically decreased the additive effect observed when U937 cells were both treated by phorbol ester and expressed the tat gene product, suggesting a high interdependence of the AP-1 and AP-4 sequences for the regulation of the transcription driven by the visna LTR. The c-Jun/AP-1 factor was a prerequisite for the modulation of the activity of the LTR since no Tat-mediated transactivation was found when transfection experiments were carried out in F9 teratocarcinoma cells which are deficient for AP-1 activity. Because the Tat product enhanced the transcription of the visna LTR via the AP-1 site, we asked whether this viral factor could regulate the expression of cellular factors involved in one of the cellular activation pathways. Northern analysis of U937 cells clearly indicated that visna Tat promoted the c-jun mRNA expression, in contrast to the c-fos mRNA expression. Next, we examined nuclear extracts prepared at various times after infection of permissive ovine cells with visna virus, and showed an increased level in the c-Jun DNA binding activity. These data indicated that viral infection can induce a cellular activation pathway in permissive cells.

Growth-related alterations induced in chick embryo fibroblasts by src-gene deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus.

Certain tdSRD viruses induce multilayered cell foci, agar colony formation, and increased density of cellular populations in line-15 chick embryo fibroblasts. These alterations in cellular growth properties are distinct from oncogenic transformation. Available evidence favors the idea that transformation-defective focus formation is caused by one or more of the replicative viral genes, but which of the three-gag, pol, or env-is involved, remains to be determined.

Characterization of visna virus mRNA.

Visna virus is a retrovirus responsible for a classical slow infection of the central nervous system of sheep. In the present work we focused our attention on the viral mRNA's. We found that, during the acute infection in vitro, (i) viral mRNA's amount to only 0.1% of the total cytoplasmic RNA, (ii) 20% of the total cytoplasmic viral RNA is found in polyribosomes, and (iii) three viral mRNA's can be identified by sucrose gradient sedimentation or polyacrylamide gel electrophoresis. Their sedimentation coefficients are 36S, 27S, and 21S.

Complexity and polyadenylic acid content of visna virus 60-70S RNA.

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.