Molecular biology of the hepatitis B virus and role of the X gene.

The hepatitis B virus (HBV) is a widespread human pathogen and a major health problem in many countries. Molecular cloning and sequencing of the viral DNA genome has demonstrated a small and compact structure organized into four overlapping reading frames that encode the viral proteins. Besides structural proteins of the core and the envelope, HBV encodes a DNA polymerase with reverse transcriptase activity, a secreted antigen of unknown function, and a transcriptional activator that is essential for viral replication. Major steps of the viral life cycle have been unraveled, including transcription of all viral RNAs from nuclear covalently closed circular DNA (cccDNA), followed by encapsidation of pregenomic RNA, a more-than-genome length transcript, and reverse transcription of pregenomic RNA leading to asymmetric synthesis of the DNA strands. Although HBV has been recognized as a human tumor virus, no direct transforming activity could be evidenced in different cellular and animal models. However, the transcriptional regulatory protein HBx encoded by the X gene is endowed with weak oncogenic activity. HBx harbors pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis.

Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1.

Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.

HTLV-I Tax and cell cycle progression.

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL) and various human myopathies/neuropathies. HTLV-I encodes a 40 kDa phosphoprotein, Tax, which has been implicated in cellular transformation. In similarity with several other oncoproteins such as Myc, Jun, and Fos, Tax is a transcriptional activator. How Tax mechanistically dysregulates the cell cycle remains unclear. Recent findings from us and others have shown that Tax targets key regulators of G1/S and M progression such as p16INK4a, cyclin D1, cyclin D3-cdk, and the mitotic spindle checkpoint apparatus. Thus, Tax influences the progression of cells in various phases of the cell cycle. In this regard, we will discuss three distinct mechanisms through which Tax affects cell-cycling: a) through direct association Tax can abrogate the inhibitory function of p16INK4a on the G1-cdks, b) Tax can also directly influence cyclin D-cdk activities by a protein-protein interaction, and c) Tax targets the HsMAD1 mitotic spindle-assembly checkpoint protein. Through these varied routes, the HTLV-I oncoprotein dysregulates cellular growth controls and engenders a proclivity of cells toward a loss of DNA-damage surveillance.

Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).

Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat.

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67: 6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.

Interaction of the second coding exon of Tat with human EF-1 delta delineates a mechanism for HIV-1-mediated shut-off of host mRNA translation.

HIV-1 Tat has pleiotropic functions. While its most studied role is to activate transcription from the retroviral long terminal repeat (LTR)-promoter, Tat also has functions as a secretable growth factor, a T-cell activator, and an inducer of cellular apoptosis, amongst others. For its transcriptional function, the first coding exon of Tat appears wholly sufficient; however, lentiviruses (HIVs and SIVs) maintain and conserve a second coding exon for Tat. While the function(s) of the second exon of Tat has remained largely unknown, its integrity in lentiviral genomes suggests biological importance, possibly a role in non-transcriptional activities. To understand better the biology of the second exon of Tat in HIV-1 infection of cells, we have searched for cellular proteins that bind specifically to this protein domain. Here, we report that the human translation elongation factor 1-delta (EF-1 delta) binds to the second exon of HIV-1 Tat. Interaction between Tat and EF-1 delta dramatically reduces the efficiency of the translation of cellular, but not viral, mRNAs. These findings suggest that a non-transcriptional activity of Tat modulates cellular protein synthesis, thereby affecting the metabolism of host cells.

Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture.

Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-Is in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tat were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections.

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.

HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors.

In 1991, we demonstrated, using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed.

Induction of NF-KB during monocyte differentiation by HIV type 1 infection.

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.

The env gene variability is not directly related to the high cytopathogenicity of an HIV1 variant.

HIV1-NDK is a Zairian HIV1 isolate which is unique because of its high cytopathic effect on T lymphoblastoid cell lines. Its sequence analysis has indicated 30% divergence with HIV1-LAV/BRU HTLVIII B prototype in the env gene encoding for the envelope glycoprotein gp120. In order to correlate the highly cytopathic properties with the env genetic variability, recombinants between the HIV1 prototype and HIV1-NDK have been constructed, including HIV1-NDK env gene, and their cytopathic phenotypes were analyzed. The viral hybrid containing all HIV1 prototype sequences except a large fragment including the total HIV1-NDK env gene remained at a low cytopathic phenotype. Our results suggest that sequences other than the env gene namely p18 gag, vif, and vpr are required for the high virulence of HIV1-NDK.

Differences in replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) are not determined by long terminal repeats (LTR).

The growth properties of molecular clones of a highly cytopathic Zairian HIV1-NDK and prototype viruses were compared to correlate genetic variations with biological changes. The cloned HIV1-NDK retained the highly replicating cytopathic phenotype and formed larger syncytia than the prototype. One of the major differences in the alignment of the nucleotide sequence of the HIV1-NDK and HIV1-BRU prototypes was localized in the negative regulatory element (NRE) of the long terminal repeat (LTR). In a chloramphenicol acetyl transferase (CAT) assay, we failed to detect a significant difference between LTR promoter activity of the prototype and HIV1-NDK, suggesting that the LTR of both phenotypes had a similar function. The complete recombinant provirus DNA molecules bearing HIV1 LTR derived from one phenotype and the rest of the genomes from the other phenotype were constructed and transfected. The high cytopathogenicity of both the original and the chimeric viruses was correlated with the high speed of virus replication. Cytopathogenicity, morphology of syncytia, and replication kinetics of the recombinant viruses were determined by the functions coded within an internal part of HIV1 genome, covering the gag to env region, which were, however, not within LTR.