Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation.

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.     

Binding of Tat protein to TAR region of human immunodeficiency virus type 1 blocks TAR-mediated activation of (2'-5')oligoadenylate synthetase.

The TAR sequence of the 5' leader of HIV-1 long terminal repeat-directed mRNA was found to be able to bind to and to activate double-stranded RNA-dependent (2'-5')A synthetase. Binding of TAR to the purified synthetase in vitro was abolished by addition of HIV-1 Tat protein, which binds to this sequence with a high affinity. Inhibition of TAR-mediated activation of (2'-5')A synthetase by Tat was prevented in the presence of the Zn2+ and Cd2+ chelators o-phenanthroline and penicillamine, which did not impair TAR-synthetase interaction. Transient expression assays of bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells revealed that the levels of both CAT mRNA and CAT protein decreased after treatment of the cells with interferon, if CAT gene was linked to HIV-1 TAR segment. Cotransfection of the cells with a tat sequence containing plasmid rendered CAT gene expression insensible to the action of interferon.     

Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.

tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.     

Relative activities of HIV-1-IIIB and HIV-1BaL LTR and tat in primary monocytes and lymphocytes

The mechanisms determining the ability of some but not other strains of human immunodeficiency virus type 1 (HIV-1) to grow in peripheral blood monocyte-macrophages are presently unclear. The tat gene of HIV-1-IIIB which replicates poorly in human macrophages, and the tat gene of HIV-1-BaL, which replicates to high titers in the same cells in transient expression systems with their respective long terminal repeats (LTR) driving a reporter chloramphenicol acetyl transferase (CAT) gene were compared. The authors hypothesized that the tat gene and LTR of BaL might help account for its efficient growth in primary monocyte-macrophages by virtue of a high activity in these cells relative to that of the IIIB tat and LTR. Primary peripheral blood lymphocytes and monocytes were cotransfected with either the HIV-1BaL or HIV-1-IIIB LTR fused to the CAT gene and their respective tat genes. The IIIB tat and LTR were at least as active in primary lymphocytes as the BaL combination, and both tat-LTR pairs were more active in primary lymphocytes than monocytes. The same relative activities were also observed in primary monocytes after in vitro maturation to macrophages prior to transfection. These data strongly suggest that neither the tat gene nor the LTR of HIV-1-IIIB and HIV-1BaL can account for the great ability of the latter or the inability of the former to grow in monocyte-macrophages.     

Human and simian immunodeficiency retroviruses: activation and differential transactivation of gene expression

New West African human immunodeficiency viruses (HIV-2s) and simian immunodeficiency virus (SIV) contain functional transactivator (tat) gene and tat response elements. Their long terminal repeats (LTR) and tat genes are more related among themselves than to HIV-1 LTR and tat gene. The viral gene expression of HIV-2 as well as SIV can be stimulated by T cell activators, such as mitogens and phorbol esters. HIV-2 and SIV display a much broader transactivation response specificity than does HIV-1. The LTR-directed gene expression of HIV-2/SIV is not only transactivated by their own tat gene and by HIV-1 tat gene but also by factors in human T cell leukemia/lymphoma virus type I (HTLV-I) and simian virus 40 (SV40) infected cells, involving HTLV-I tat gene and SV40 T antigens, respectively. HIV-1 LTR-directed gene expression is much less transactivated by HIV-2/SIV tat genes and by factors in HTLV-I- and SV40-infected cells. Immune activation and heterologous transactivation of the LTR-directed gene expression may be relevant to the latency of virus infection and progression toward the acquired immunodeficiency syndrome (AIDS).     

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression.

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.     

Cytokine augmentation of HIV-1 LTR-driven gene expression in neural cells.

The induction of human immunodeficiency virus type 1 (HIV-1) gene expression by cytokines was investigated in cells of central nervous system origin. These were human neuroblastoma, glioblastoma, and astrocytoma cell lines, a murine oligodendroglioma and primary murine astrocyte cultures. The cytokines used were tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), IL-6, and interferons alpha and gamma (IFN alpha, gamma). Transient transfection of cells with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) showed significant augmentation following treatment by particular cytokines. TNF alpha was found to augment HIV LTR-directed CAT activity in all cell types. IL-1 beta also activated the HIV LTR reporter gene in glioblastoma, astrocytoma, and astrocyte cells. IL-6 enhanced HIV gene expression in one example only, the primary astrocyte cultures. The interferons generally suppressed expression from the LTR except IFN gamma which produced a twofold rise in the murine glial cells and IFN alpha augmenting expression in one neuroblastoma cell line. No synergy was observed between pairs of activating cytokines tested. The HIV tat gene product was found to be functional in all cells, cotransfection of a tat expression vector transactivating expression from the LTR, with varying degrees of efficiency. In some cell lines the combination of an activating cytokine and tat resulted in an enhancement above that obtained by cotransfection of tat alone. In others, the level of CAT activity did not significantly change. Analysis of nuclear extracts from cytokine-treated cells further implicated the involvement of NFKB in the induction of HIV-1 gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses.

To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.     

Effect of the calcium channel blocker verapamil on human immunodeficiency virus type 1 replication in lymphoid cells.

Cell signaling events are known to affect human immunodeficiency virus type 1 (HIV-1) replication. Treatment of lymphoid CEM cells with the calcium channel blocker verapamil (25-75 microM) enhanced HIV-1 expression in acute, whole virus infection experiments, despite lowering intracellular calcium levels, ablating the acute rise in intracellular calcium normally seen with infection, and lengthening the doubling time of cell replication. Verapamil had no effect on cell surface CD4 expression. Transfection of CEM cells with plasmids containing the HIV-1 long terminal repeat linked to the chloramphenicol acetyltransferase reporter gene showed that verapamil enhanced expression of the HIV-1 long terminal repeat in a dose-dependent fashion. This effect was abolished by mutations in the binding sites for nuclear factor kappa-B. Electrophoretic mobility shift assays confirmed that verapamil induced nuclear factor kappa-B activity in CEM cells. Thus, verapamil, in high concentrations, can potentiate HIV-1 replication in lymphoid cells, and this effect may be mediated by induction of nuclear factor kappa-B.