Search

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379-654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis.

A mini‐F region 800 bp long, located between the two F origin sites, plays an essential role in the relationship between the F plasmid and its host. This region comprises two sets of overlapping coding sequences: the first set codes for the newly identified H1 and H2 polypeptides; the second set codes for polypeptides G1 and G2. A mini‐F amber mutation (Ham22) causes the virtual disappearance of polypeptides H1 and H2 but only slightly reduces synthesis of polypeptides G1 and G2. This mutation: (i) renders mini‐F hybrids lethal to the host cells (conditional Hos‐ phenotype for host survival) and (ii) causes the induction of a resident prophage in recA+ strains (conditional Map‐ phenotype for maintenance of the prophage). When an additional mutation prevents the synthesis of polypeptides G1 and G2, both the lethal character and the induction of the prophage are abolished. We conclude: (i) that polypeptides G1 and/or G2 are specific mini‐F polypeptides involved in the plasmid‐mediated killing effect and in the recA‐dependent induction of the resident prophage and (ii) that, in normal conditions, polypeptides H1 and/or H2 negatively control (directly or indirectly) the action of polypeptides G1 and/or G2. In relation to the analysis of indirect induction mediated by u.v.‐irradiated lambda mini‐F hybrids, we propose that polypeptides G1 and/or G2 are specific mini‐F products involved in the activation of the bacterial SOS pathway. The H1/H2 and G1/G2 polypeptides could constitute the controlled mini‐F signal enabling the coordination between cell division and F plasmid replication.

The elements which ensure the maintenance of the F plasmid are located in its f5 EcoRI restriction fragment. This f5 fragment constitutes a mini‐F plasmid showing the same stability and copy number control as the entire F plasmid. The proteins expressed in minicells by wild‐type or mutated f5 fragments were analysed by pH gradient two‐dimensional electrophoresis. We identified seven f5‐encoded polypeptides and located their genes on the F map. Among them, H1, an acidic polypeptide of mol. wt. 10.5 K, had not been detected before. It is in fact the most abundant f5‐encoded polypeptide identified so far. In addition, we showed that both 10.5‐K and 12‐K protein bands detected by SDS‐polyacrylamide gel electrophoresis are, respectively, composed of two polypeptides, H1 and H2, G1 and G2, of different isoelectric points. Polypeptides H2 and G2, respectively, share common coding sequences with polypeptides H1 and G1. Their possible biological significance is discussed. The sequences coding for polypeptides H1/H2 and G1/G2 are clustered in a 800‐bp long region located between the two mini‐F origin sites and are proposed to be organized as an operon. The results reported in the accompanying paper point out the importance of polypeptides G1/G2 and H1/H2 in the relationship between the F plasmid and its host.

Immunotherapy by adoptive transfer of lymphocytes was attempted in identical twins, one who was virus-free and the other who was infected with human immunodeficiency virus-1 (HIV-1), at the stage of acquired immunodeficiency syndrome. The noninfected twin was vaccinated by priming with a recombinant vaccinia virus expressing the envelope glycoprotein of one of his brother’s viruses and boosting with the same purified gp160 adsorbed on alum. Vaccination elicited major histocompatibility complex class I-restricted CD8+cytolytic T lymphocytes specific for HIV-1, but no antibody response. The diseased brother, a 38-year-old homosexual who had developed repeated opportunistic infections since 1990 and had a CD4+count reduced to practically zero, was treated by infusions of lymphocytes collected from the vaccinated brother by lymphopheresis. After a first transfer of the whole lymphocyte population, no changes were observed in the clinical status and biologic or virologic parameters. A second transfer was then applied with activation of the cells with purified envelope glycoprotein before infusion. The outcome of the treatment was an increase in total lymphocytes, in CD4+and activated CD8+DR+cell counts, and in proliferative responses to HIV antigens. A marked but transient 3-log increase in cellular and plasmatic virus loads was also observed after the second adoptive transfer. These observations will be considered with attention to improve the future adoptive transfer protocols, especially in patients with severe CD4+depletion.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N‐terminal glycine codon (Gly‐‐‐‐Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100‐120 nm virus‐like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

Adult prooestrous rats were injected subcutaneously with chlorpromazine prior to the »critical period« (2:00 p. m.–4:00 p.m.) to prevent ovulation. Midway through the »critical period« (3:00 p. m.) these blocked females were injected intravenously with varying doses of follicle stimulating hormone which had been maintained overnight at room temperature or incubated with chymotrypsin overnight in a waterbath maintained between 35–36°C. Results showed that the amount of NIHFSH-S7 calculated to cause ovulation was nearly identical for enzyme exposed (15.49 μg) and unexposed (15.36 μg) FSH.The Parlow assay was used to verify that the enzyme specifically digested the LH contaminant of FSH. Significant depletions of ascorbic acid were obtained only with NIHFSH-S7 samples that were unexposed to the enzyme. Overnight incubation of FSH with chymotrypsin completely prevented depletion of ovarian ascorbic acid, verifying that the enzyme digested the LH contaminant present in NIHFSH-S7. These data further support our concept that follicle stimulating hormone by itself induces ovulation in CPZ blocked rats.