Retroviral cyclin controls cyclin-dependent kinase 8-mediated transcription elongation and reinitiation.

UNLABELLED: Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs]) FOS, EGR1, and cJUN. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RV-cyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the EGR1 gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended EGR1 gene expression. The RV-cyclin increases CDK8 occupancy at the EGR1 gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis. IMPORTANCE: The data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in many human cancers. CDK8 controls expression from highly regulated genes, including the interferon-stimulated genes. Its function is likely the target of many viral interferon-resistance mechanisms. CDK8 also controls cellular responses to metabolic stimuli, stress, and hypoxia, in addition to the serum response. The retroviral cyclin (RV-cyclin) represents a highly selected probe of CDK8 function. RV-cyclin does not control CDK8 specificity but instead enhances CDK8's effects on regulated genes, an important distinction for its use to delineate natural CDK8 targets. The outcomes of this research are applicable to investigations of normal and abnormal CDK8 functions. The mechanisms defined here will contribute directly to the dermal sarcoma model in fish and clarify an important path for oncogenesis and innate resistance to viruses.

Charge-surrounded pockets and electrostatic interactions with small ions modulate the activity of retroviral fusion proteins.

Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.

Requirement of the human T-cell leukemia virus p12 and p30 products for infectivity of human dendritic cells and macaques but not rabbits.

The identification of the genes necessary for human T-cell leukemia virus (HTLV-1) persistence in humans may provide targets for therapeutic approaches. We demonstrate that ablation of the HTLV-1 genes encoding p12, p30, or the HBZ protein, does not affect viral infectivity in rabbits and in this species, only the absence of HBZ is associated with a consistent reduction in virus levels. We observed reversion of the HTLV-1 mutants to the HTLV-1 wild-type genotype in none of the inoculated rabbits. In contrast, in macaques, the absence of HBZ was associated with reversion of the mutant virus to the wild-type genotype in 3 of the 4 animals within weeks from infection. Similarly, reversion to the wild type was observed in 2 of the 4 macaque inoculated with the p30 mutant. The 4 macaques exposed to the p12 knock remained seronegative, and only 2 animals were positive at a single time point for viral DNA in tissues. Interestingly, we found that the p12 and the p30 mutants were also severely impaired in their ability to replicate in human dendritic cells. These data suggest that infection of dendritic cells may be required for the establishment and maintenance of HTLV-1 infection in primate species.

Retroviral integrase superfamily: the structural perspective.

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid-processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA-mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus--RNase H1 and integrase--are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.

[Virological aspects of HTLV-1 infection and new therapeutical concepts]. / Aspects virologiques de l'infection par HTLV-1 et nouveaux concepts thérapeutiques.

HTLV-1 was the first human oncogenic retrovirus to be discovered. It is the etiological agent of adult T leukemia/lymphoma (ATLL) and of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), two diseases that develop after a long latency period. Importantly, HTLV-1 does not cause ATLL through insertional mutagenesis. Apart from the gag, pro, pol and env genes, which are common to all retroviruses, HTLV-1 genome also encodes regulatory and auxiliary viral proteins. Among the former, Tax promotes cell transformation and HBZ is involved in the leukemic cells proliferation and in the maintenance of the transformed phenotype. Anti-ATLL therapies have lately made significant progress with an efficient antiviral treatment against the chronic and smoldering forms of this leukemia, but an efficient treatment of TSP/HAM patients is still lacking. Results from a recent study associating histone acetylase inhibitor with an anti-viral drug will be discussed here. While an increase in proviral load is considered a marker for disease progression, this treatment allows a significant drop of the proviral load in asymptomatic carriers.

The endogenous mink cell focus-forming (MCF) gp70 linked to the Rmcf gene restricts MCF virus replication in vivo and provides partial resistance to erythroleukemia induced by Friend murine leukemia virus.

The Rmcf locus restricts the in vitro replication of mink cell focus-forming (MCF) viruses in cell cultures derived from mice carrying the resistance allele. Previously we reported that in cell cultures from first backcross progeny, this Rmcf-linked restriction segregates with the expression of an endogenous retroviral gp70 serologically related to that of MCF viruses. The current report details the results of genetic studies designed to examine the possible association of this endogenous gp70 with resistance of mice to Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. This env gene segregates as a single dominant trait in (DBA/2 X IRW) X IRW progeny, in which the expression of the gene can be detected by serological techniques. Results indicated that the gp70- progeny developed leukemia at the same rate as the susceptible IRW parent, whereas the tempo of disease among the gp70+ progeny was significantly slower. However, the resistance mediated by this gene was only partial, since most of the gp70+ offspring eventually developed erythroleukemia when followed for 6 mo. This endogenous gp70 also segregated with a restriction to the expression of recombinant MCF viruses after infection with F-MuLV. Since in this study all unlinked genes segregated independently, this is direct evidence that MCF viruses participate in the induction of erythroleukemia.

Simian immunodeficiency virus SIVagm from African green monkeys does not antagonize endogenous levels of African green monkey tetherin/BST-2.

The Vpu accessory gene that originated in the primate lentiviral lineage leading to human immunodeficiency virus type 1 is an antagonist of human tetherin/BST-2 restriction. Most other primate lentivirus lineages, including the lineage represented by simian immunodeficiency virus SIVagm from African green monkeys (AGMs), do not encode Vpu. While some primate lineages encode gene products other than Vpu that overcome tetherin/BST-2, we find that SIVagm does not antagonize physiologically relevant levels of AGM tetherin/BST-2. AGM tetherin/BST-2 can be induced by low levels of type I interferon and can potently restrict two independent strains of SIVagm. Although SIVagm Nef had an effect at low levels of AGM tetherin/BST-2, simian immunodeficiency virus SIVmus Vpu, from a virus that infects the related monkey Cercopithecus cephus, is able to antagonize even at high levels of AGM tetherin/BST-2 restriction. We propose that since the replication of SIVagm does not induce interferon production in vivo, tetherin/BST-2 is not induced, and therefore, SIVagm does not need Vpu. This suggests that primate lentiviruses evolve tetherin antagonists such as Vpu or Nef only if they encounter tetherin during the typical course of natural infection.

Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR.

In studies of the genetics of human immunodeficiency virus type 1 (HIV-1), the product of the nef gene, formerly known as F, 3'-orf, or B-ORF, was a negative regulator of HIV-1 replication. Proviruses with mutations in the nef gene replicated better than their standard counterparts during transient expression, and the mutant virus maintained its enhanced replication even after serial passages in T lymphocytes. The nef protein trans-suppressed, in a dose-dependent manner, the replication of wild-type and nef mutant proviruses and the expression of reporter genes linked to the HIV-1 long terminal repeat (LTR). The repression induced by the nef protein was mediated by inhibition of transcription from the HIV-1 LTR, which contains a far upstream cis element (previously recognized to be a negative regulatory element) between 340 and 156 nucleotides upstream of the RNA initiation site.

Transformation by v-sis occurs by an internal autoactivation mechanism.

Transformation by the v-sis oncogene appears to require an interaction of its protein product, p28v-sis, with the receptor for the platelet-derived growth factor (PDGF). However, this interaction may not occur at the cell surface as predicted by the autocrine hypothesis because phenotypic transformation was not reversed by incubation of SSV-NRK cells with antisera to PDGF and because morphological transformation did not occur when nontransformed NRK cells were cultured continuously with p28v-sis. A mutant of the wild-type v-sis gene was constructed that encodes a v-sis protein targeted for retention within the endoplasmic reticulum and Golgi. NRK cells expressing the mutant v-sis gene did not secrete any detectable v-sis protein but were as fully transformed as wild-type v-sis transfectants. The results support a mechanism of transformation by v-sis in which internal activation of the PDGF receptor occurs before expression of either p28v-sis or the PDGF receptor at the cell surface.

Fibroblasts transformed with v-src show enhanced formation of an inositol tetrakisphosphate.

The tyrosine kinase pp60v-src, encoded by the v-src oncogene, seems to regulate phosphatidylinositol metabolism. The effect of pp60v-src on control points in inositol phosphate production was examined by measuring the amounts of inositol polyphosphates in Rat-1 cells expressing wild-type or mutant forms of the protein. Expression of v-src-resulted in a five- to sevenfold elevation in the steady-state amount of an isomer of inositol tetrakisphosphate, whereas the concentrations of inositol trisphosphates or other inositol tetrakisphosphates were not affected. The activity of a key enzyme in the formation of inositol tetrakisphosphates, inositol (1,4,5)-trisphosphate 3-kinase, was increased six- to eightfold in cytosolic extracts prepared from the v-src-transformed cells, suggesting that this enzyme may be one target for the pp60v-src kinase and that it may participate in the synthesis of novel, higher order inositol phosphates.

The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts.

The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) is known to efficiently transform NIH/3T3 fibroblasts in vitro and to cause an acute lymphosarcoma in susceptible murine hosts. The role of its relative, the bcr/abl gene product, in the etiology of human chronic myelogenous leukemia (CML) remains speculative. To assess the transforming properties of the bcr/abl gene product, complementary DNA clones encoding the CML-specific P210 bcr/abl protein were expressed in NIH/3T3 fibroblasts. In contrast to the v-abl oncogene product P160, the P210 bcr/abl gene product did not transform NIH/3T3 cells. Cell lines were isolated that expressed high levels of the P210 bcr/abl protein but were morphologically normal. During the course of these experiments, a transforming recombinant of bcr/abl was isolated which fuses gag determinants derived from helper virus to the NH2-terminus of the bcr/abl protein. This suggests that a property of viral gag sequences, probably myristylation-dependent membrane localization, must be provided to bcr/abl for it to transform fibroblasts.

HTLV-I tax induces cellular proteins that activate the kappa B element in the IL-2 receptor alpha gene.

Jurkat T cell lines constitutively expressing Tax, the 40-kilodalton transactivator protein of human T lymphotropic virus type I (HTLV-I), were used to investigate the mechanism by which this viral product deregulates the expression of the interleukin-2 receptor alpha gene (IL-2R alpha, Tac). Transfection of deleted forms of the IL-2R alpha promoter and in vitro DNA-binding studies revealed that a 12-base pair promoter segment, which has homology with the binding site for NF-kappa B, was required for Tax-induced activation of the IL-2R alpha promoter in vivo. An 18-base pair oligonucleotide containing this kappa B-like regulatory element proved sufficient to confer Tax inducibility upon a heterologous promoter. DNA affinity precipitation assays showed that Tax, like mitogenic stimuli, induced the expression of the 86-kilodalton cellular protein HIVEN86A, which specifically binds to the IL-2R alpha kappa B element in vitro. Furthermore, DNA/protein cross-linking studies revealed that several polypeptides interact with this sequence motif. Thus, the deregulation of IL-2R alpha gene expression encountered in HTLV-I leukemias appears to involve Tax activation of one or more cellular proteins that are normally induced by mitogens and that directly contribute to transcriptional activation of this receptor gene.

Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization.

Rabbit antisera were raised against three overlapping synthetic peptides with sequence homology to the second conserved domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV). All of the antisera immunoprecipitated the envelope glycoprotein. In particular, an antiserum directed against amino acids 254 to 274 of env was efficient in neutralizing three different isolates of HIV in vitro, without affecting the binding of the virus to CD4-positive cells. Therefore, this conserved region of gp120 appears to be critical in a postbinding event during virus penetration and may represent a target for antibody neutralization of HIV. These findings may be applicable in the design of a vaccine for the acquired immunodeficiency syndrome.

Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I.

To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation.

Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells.

The first step in the infection of human T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is attachment to the target cell receptor, the CD4 antigen. This step may be vulnerable to attack by antibodies, chemicals, or small peptides. Dextran sulfate (molecular weight approximately 8000), which has been given to patients as an anticoagulant or antilipemic agent for more than two decades, was found to block the binding of virions to various target T lymphocytes, inhibit syncytia formation, and exert a potent inhibitory effect against HIV-1 in vitro at concentrations that may be clinically attainable in human beings. This drug also suppressed the replication of HIV-2 in vitro. These observations could have theoretical and clinical implications in the strategy to develop drugs against HIV types 1 and 2.

Murine endogenous retroviruses.

Up to 10% of the mouse genome is comprised of endogenous retrovirus (ERV) sequences, and most represent the remains of ancient germ line infections. Our knowledge of the three distinct classes of ERVs is inversely correlated with their copy number, and their characterization has benefited from the availability of divergent wild mouse species and subspecies, and from ongoing analysis of the Mus genome sequence. In contrast to human ERVs, which are nearly all extinct, active mouse ERVs can still be found in all three ERV classes. The distribution and diversity of ERVs has been shaped by host-virus interactions over the course of evolution, but ERVs have also been pivotal in shaping the mouse genome by altering host genes through insertional mutagenesis, by adding novel regulatory and coding sequences, and by their co-option by host cells as retroviral resistance genes. We review mechanisms by which an adaptive coexistence has evolved. (Part of a multi-author review).

HTLV-1 and apoptosis: role in cellular transformation and recent advances in therapeutic approaches.

A universal cellular defense mechanism against viral invasion is the elimination of infected cells through apoptotic cell death. To counteract host defenses many viruses have evolved complex apoptosis evasion strategies. The oncogenic human retrovirus HTLV-1 is the etiological agent of adult-T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disease known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The poor prognosis in HTLV-1-induced ATLL is linked to the resistance of neoplastic T cells against conventional therapies and the immuno-compromised state of patients. Nevertheless, several studies have shown that the apoptotic pathway is largely intact and can be reactivated in ATLL tumor cells to induce specific killing. A better understanding of the molecular mechanisms employed by HTLV-1 to counteract cellular death pathways remains an important challenge for future therapies and the treatment of HTLV-1-associated diseases.

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T Cell Leukemia/Lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.

v-src inhibits differentiation via an extracellular intermediate(s).

Murine 3T3L1 preadipocytes transformed by avian sarcoma virus were unable to differentiate in response to insulin or dexamethasone plus 1-methyl-3-isobutylxanthine, both potent inducers of differentiation of the nontransformed 3T3L1 parental line. Conditioned medium from transformed cells contained a relatively heat-stable factor(s) which inhibited the differentiation of untransformed parental 3T3L1 cells but did not induce any changes in their morphology. A protease-sensitive mitogen was also detected in the medium. The relationship between the two activities remains to be elucidated.