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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.

Abstract.: The effects of OST-766, an inhibitor of vacuolar H+-ATPase activity, on adenylyl cyclase and phospholipase C activity were explored in the osteoblast cell line ROS 17/2.8. In fresh homogenates of ROS 17/2.8 cells, OST-766 inhibited adenylyl cyclase activity (ACA) in response to guanine nucleotide and forskolin but had no effect on basal ACA. OST-766 enhanced the basal generation of IP2, but not that formed in response to Ca2+ or guanine nucleotides. In marked contrast, incubation of intact ROS 17/2.8 cells with OST-766 for at least 48 hours resulted in an increase in basal ACA as well as in response to PTH, guanine nucleotides and forskolin. Under similar conditions, the compound also increased IP1, IP2 and IP3 generation in response to guanine nucleotides and Ca2+. Levels of the guanine nucleotide binding proteins Gs and Gi were also increased in OST-766-treated cells. The results suggest that the actions of this H+-ATPase inhibitor include effects on osteoblasts through PTH-sensitive signal transduction pathways.

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.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.

Mills KHG, Page M, Chan WL, Kitchin P, Stott EJ, Taffs F, Jones W, Rose J, Ling C, Silvera P, Corcoran T, Flanagan B, Burny A, Bex F, Delchambre M, Van Opstal O, Fabry L, Thiriart C, Delers A, DeWilde M, Bruck C. Protection against SIV infection in macaques by immunization with inactivated virus from the BK28 molecular clone, but not with BK28‐derived recombinant env and gag proteins. J Med Primatol 1992:21:50‐58. Vaccination of cynomolgus macaques with β‐propiolactone inactivated SIVmacBK28in Freund's adjuvant induced low but detectable levels of anti‐SIV envelope (env) antibodies and T‐cell responses and protected against challenge with the 32H isolate of SIVmac251grown in C8166 cells. In contrast, purified recombinant SIV env and gag proteins derived from BK28 formulated in Syntex adjuvant generated consistent and long‐lived cellular and humoral immune responses to SIV env, but failed to protect against infection with the 32H virus. Thus, protection against a heterogeneous challenge stock is possible by immunization with a molecularly‐cloned virus, but not with recombinant proteins from the same molecular origin. High levels of anti‐cell antibodies induced by the whole virus vaccine, but not by recombinant proteins, may have contributed to the protection observed.

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.

Cytotoxic T lymphocytes (CTL) may be important to prevent cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), the agent responsible for AIDS. In this study, we investigated the epitope specificity of CTLs induced in individuals immunized against the virus envelope glycoprotein gp160. The determinant of HIV-1 gp160 for the stimulation of CTL is located in a region of high sequence variability among HIV-1 isolates, the so-called V3 loop P18. Using a panel of P18 peptides, we compared the CTL specificities of cells from two individuals immunized with vaccinia virus recombinants expressing the envelope glycoproteins from two different strains of HIV-1, IIIB and SIMI. For this purpose, CTLs specific for the IIIB P18 peptide (RIQRGPGRAFVTIGK) were compared with CTLs for the site from the SIMI isolate (TLHMGPKRAFYATGD). The results indicate that in contrast to CD8+ CTLs induced by the glycoprotein from strain IIIB, CD8+ CTLs induced by strain SIMI strongly cross-reacted with targets presenting P18 peptides as well as envelope proteins from the divergent MN and RF isolates but failed to cross-react with targets that presented the IIIB peptide. These data have implications for the design of an HIV vaccine.

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.

Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14-bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus-challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregulatory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vif gene and first coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses.

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.

The fusion domain of simian immunodeficiency virus (SIV) envelope glycoproteins is a hydrophobic region located at the amino‐terminal extremity of the transmembrane protein (gp32). Assuming an alpha helical structure for the SIV fusogenic domain of gp32 in a lipid environment, theoretical studies have predicted that the fusion peptide would insert obliquely in the lipid bilayer. This oblique insertion could be an initial step of the fusion process by disorganizing locally the structure of the lipid bilayer. We have tested this hypothesis by selectively mutagenizing the SIV gp160 expressed via a vaccinia virus vector, to alter the theoretical angle of insertion of the fusion peptide. The fusogenic activity of the wild‐type and mutant glycoproteins was tested after infection of T4 lymphocytic cell lines by the recombinant vaccinia virus, and measure of syncytia formation. Mutations that modified the oblique orientation reduced the fusogenic activity. In contrast, mutations that conserve the oblique orientation did not alter the fusogenic properties. Our results support the hypothesis that oblique orientation is important for fusogenic activity.

In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Apr plasmid: pSC138 (Timmis et al., 1975). ?F recombinants were shown to be chimerae between the ? and the F replicons. By genetical tests, we have demonstrated that both ? and F replication mechanisms are functional: the ?F recombinant behaves as a non defective plaque forming phage on ? sensitive bacteria and establishes itself as a stable plasmid on recA F- homoimmune bacteria. In the extra-chromosomal state, the ?F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.

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.

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, U.S.A.(Received2% March1978)Tamoxifen (trans1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene; ICI 46,474) is both a potent antioestrogen and a weak oestrogen (Harper & Walpole, 1967) and is thought to act by inhibiting the binding of oestradiol to its cytoplasmic receptor (Jordan & Koerner, 1975). Chronic treatment with tamoxifen causes atrophy of the male accessory reproductive glands and the testicular germinal epithelium in the rat (Harper & Walpole, 1967) and the dog (M. Mason, unpublished data), as well as depression of the plasma concentration of testosterone in the latter species (M. Mason & A. Bartke, unpublished data). A direct inhibitory effect of tamoxifen on testicular steroidogenesis was suspected because treatment of female rats with tamoxifen increases, rather than decreases, the plasma concentration of luteinizing hormone (LH; Watson, Anderson, Alam, O'Grady & Heald, 1975). The possibility that an antioestrogen might act directly is of interest since

In prolactin (PRL)-deficient male dwarf mice, treatment with PRL stimulates the release of FSH without affecting plasma LH levels. We now report that this effect of PRL is not mediated by the testes and that PRL does not modify FSH or LH release in female dwarf mice.

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