Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells.

The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. Two subtypes of human T lymphotropic virus (HTLV) are associated with different disease phenotypes. In this article it is shown that marked differences exist in the ability of LTR sequences of these subtypes to function as transcriptional elements in differentiated cell types. It is also shown that trans-acting regulatory factors present in HTLV-infected cells stimulate gene expression directed by these LTR sequences in a type-specific manner. These results have implications for understanding the diverse biological effects of HTLV infection.

Trans activation of the bovine leukemia virus long terminal repeat in BLV-infected cells.

The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. This study shows that factors present in cells infected with bovine leukemia virus (BLV) mediate transcriptional trans activation of the BLV LTR. This phenomenon is similar to that reported for the human T-cell leukemia virus LTR and establishes both structural and functional criteria for inclusion of BLV and human T-cell leukemia viruses in the same family of transforming retroviruses.

Trans-activator gene of HTLV-II induces IL-2 receptor and IL-2 cellular gene expression.

The human T-lymphotropic viruses types I and II (HTLV-I and -II) have been etiologically linked with certain T-cell leukemias and lymphomas that characteristically display membrane receptors for interleukin-2. The relation of these viruses to this growth factor receptor has remained unexplained. It is demonstrated here that introduction of the trans-activator (tat) gene of HTLV-II into the Jurkat T-lymphoid cell line results in the induction of both interleukin-2 receptor and interleukin-2 gene expression. The coexpression of these cellular genes may play a role in the altering T-cell growth following retroviral infection.

Antigens encoded by the 3'-terminal region of human T-cell leukemia virus: evidence for a functional gene.

Antibodies in sera from patients with adult T-cell leukemia-lymphoma or from healthy carriers of type I human T-cell leukemia virus (HTLV) recognize an antigen of approximately 42 kilodaltons (p42) in cell lines infected with HTLV-I. Radiolabel sequence analysis of cyanogen bromide fragments of p42 led to the conclusion that this antigen is encoded in part by LOR, a conserved portion of the "X" region that is flanked by the envelope gene and the 3' long terminal repeat of HTLV-I. It is possible that this novel product mediates the unique transformation properties of the HTLV family.

Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III.

Antibodies from the serum of patients with the acquired immune deficiency syndrome (AIDS) or with the AIDS-related complex and from the serum of seropositive healthy homosexuals, recognize two major glycoproteins in cells infected with human T-cell lymphotropic virus type III (HTLV III). These glycoproteins, gp160 and gp120, are encoded by the 2.5-kilobase open reading frame located in the 3' end of the HTLV-III genome, as determined by amino terminus sequence analysis of the radiolabeled forms of these proteins. It is hypothesized that gp160 and gp120 represent the major species of virus-encoded envelope gene products for HTLV-III.

Constitutive expression of HIV-1 tat protein in human Jurkat T cells using a BK virus vector.

The production and characterization of Jurkat cell lines that constitutively express functional human immune deficiency virus type 1 (HIV-1) tat protein, using a BK virus plasmid expression vector and HIV-1 tat cDNA, is described. An increased growth rate of these Jurkat-tat cell lines as compared with control cell lines was observed.

p21rex protein of HTLV-1.

Virion protein expression of the human T-cell leukemia virus type 1 (HTLV-1) is regulated by p27rex. This protein is expressed by a doubly spliced mRNA encoding an additional protein, p21rex. p27rex has been shown to increase the amount of unspliced viral mRNAs encoding envelope and capsid proteins. To assess a possible contribution of p21rex to the rex regulation, a point mutation was introduced into the p21rex initiation codon. Expression of env and gag plasmids, transfected in Jurkat cells, was dependent only upon p27rex expression and was not affected by the absence of p21rex. Consequently, the rex regulation of the HTLV-1 structural genes can be solely attributed to the p27rex protein.

Identification of HIV-1 vpr product and function.

To investigate the role of vpr (viral protein R) in the replication and cytopathicity of human immunodeficiency virus type 1 (HIV-1), infectious proviruses were constructed that were isogenic except for the ability to produce the protein product of vpr. The experiments described here demonstrate that vpr encodes a 96 amino acid 15 kDa protein. The vpr product increases the rate of replication and accelerates the cytopathic effect of the virus in T cells. Vpr acts in trans to increase levels of viral protein expression. The stimulatory effect of vpr is observed to act on the HIV-1 LTR as well as on several heterologous promoters.

The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation.

The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B. The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but failed to respond to these mitogenic stimuli if both sequences were absent. The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells. Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester.

Cis-acting sequences responsive to the rev gene product of the human immunodeficiency virus.

Expression of high levels of the structural proteins of the human immunodeficiency virus type 1 (HIV-1) requires the presence of two regulatory genes, the trans-activator (tat), and the regulator of virion protein expression (rev. previously called art or trs). The experiments described here show that expression of virion proteins is dependent upon a small region located in the envelope gene called the cis-acting antirepression sequence (CAR). The CAR region of the envelope sequence is both necessary and sufficient for rev-dependent capsid protein expression. The experiments also show that a defect in either rev or CAR results in a dramatic decrease in the accumulation of the genomic and envelope mRNAs and an overproduction of more extensively spliced viral mRNA species.

The T open reading frame of human immunodeficiency virus type 1.

Sequence analysis of multiple isolates of the human immunodeficiency virus type 1 (HIV-1) reveals the existence of a conserved open reading frame, designated T, that partially overlaps the tat, rev, and vpu coding sequences. Here we show that in vitro translation of RNA derived from this region of the viral genome yields a 17 kDa fusion protein, the result of a minus one frameshift event in the overlap between the tat and T open reading frames. It is also shown that messenger RNA species accumulate in HIV-1 infection from which the 17 kDa protein can be made. These observations suggest that ribosomal frameshift events may result in the biosynthesis of viral regulatory as well as viral structural proteins.

Synthesis of the 2-chloro analogues of 3'-deoxyadenosine, 2',3'-dideoxyadenosine, and 2',3'-didehydro-2',3'-dideoxyadenosine as potential antiviral agents.

2-Chloro-3'-deoxyadenosine (2-chlorocordycepin), 2-chloro-2',3'-dideoxyadenosine (2-ClddAdo), and 2-chloro-2',3'-didehydro-2',3'-dideoxyadenosine (2-ClddeAdo) were synthesized from 2-chloroadenosine (2-ClAdo) as candidate antiretroviral agents on the basis that 2-chloro substitution would prevent enzymatic deamination and increase efficacy relative to 2',3'-dideoxyadenosine (ddAdo). Reduction of 2-chloro-5'-(4,4'-dimethoxytrityl)-2',3'-O-thiocarbonyladenosine with n-Bu3SnH, followed by detritylation with AcOH, unexpectedly gave a mixture of 2-chlorocordycepin and 2-chloroadenine. Treatment of the crude n-Bu3SnH reduction product with 1,1'-thiocarbonyldiimidazole, followed by another cycle of n-Bu3SnH reduction and detritylation with silica gel afforded 2-ClddAdo and a byproduct identified as 2-chloro-2',3'-O-methyleneadenosine. Treatment of 2-chloro-5'-O-(4,4'-dimethoxytrityl)-2',3'-thiocarbonyladenosine with 1,3-dimethyl-2-phenyl-1,3,2-diazaphospholidine followed by silica gel detritylation afforded 2-ClddeAdo. 2-ClddAdo and 2-ClddeAdo were tested for activity against human immunodeficiency virus (HIV) in a cultured human T4+ lymphocyte cell line. At a concentration of 100 microM, 2-ClddAdo inhibited reverse transcriptase (RT) production by 97%, while 2',3'-dideoxyadenosine (ddAdo) gave greater than 99% inhibition. In growth assays against uninfected T4+ cells, however, 100 microM 2-ClddAdo gave 23% inhibition while 100 microM ddAdo was nontoxic. At a nontoxic concentration of 20 microM, RT production was 75% inhibited by ddAdo but only 43% inhibited by 2-ClddAdo. Thus, a 2-chloro substituent increased host cell toxicity but decreased antiretroviral activity. The unsaturated analogue 2-ClddeAdo was more cytotoxic than 2-ClddAdo, and antiviral effects could not be measured above 20 microM, where there was only 75% inhibition of RT production. Because of the decreased therapeutic index of 2-ClddeAdo relative to 2-ClddAdo and ddAdo, greater than 90% inhibition of viral protein synthesis at a noncytotoxic concentration could not be achieved. In growth assays with cultured human T and B lymphocytes, 100 microM 2-chlorocordycepin gave 60-70% growth inhibition, while the IC50 against mouse fibroblasts was only 30 microM. The high cytotoxicity of 2-chlorocordycepin precluded consideration of this compound as an antiviral agent.

Cyclic AMP-mediated growth arrest is associated with increased expression of human T cell leukemia virus type I structural and transforming genes.

The effect of increased intracellular cyclic AMP levels on gene expression of the human T cell leukemia virus type I (HTLV-I) provirus was examined. Induction of infected cells to produce elevated levels of cyclic AMP was associated with specific increases in viral surface antigen expression, protein synthesis, p24 release into the supernatant, and RNA levels. The patterns of HTLV-I proviral gene expression observed support results from transfection experiments regarding the function of Tax, Rex, and cyclic AMP in HTLV-I gene regulation. As evidenced by thymidine incorporation, treatment of the infected cells to produce cyclic AMP caused reversible growth arrest. The data indicate that HTLV-I RNA and protein synthesis can proceed at an elevated level in the absence of cell growth. Sustained increases in the intracellular level of cyclic AMP may represent a method for enriching cell cultures in HTLV-I proteins.

Transcription directed by the HIV long terminal repeat in vitro.

The long terminal repeat (LTR) of the AIDS virus (HIV) has been found to contain promoter sequences that are active in uninfected HeLa whole cell and nuclear extracts. Here we report that elements upstream of position -104 (start site +1) do not affect transcriptional activity in vitro whereas sequences between -104 and -57 are required for such activity. Using a reconstituted RNA polymerase II system, we demonstrate that a partially purified fraction containing Spl not only stimulates, as was previously reported, but is required for accurate initiation of transcription directed by the HIV LTR. In addition, based on a computerized analysis, we report the presence of a region in the HIV LTR (positions -151 to -80) that is similar to the 72 base pair enhancer element of SV40 and that includes a highly conserved segment also present in the cytomegalovirus enhancer. Moreover, the HIV and HTLV-I LTRs are shown to share a region of similarity that includes the 21 base pair motif found in the enhancers of the human and bovine T-lymphotropic viruses. The R region of the HIV LTR is found to have two extensive regions of dyad symmetry rather than one as was previously reported. The significance of these observations for HIV pathogenesis is discussed.

Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization.

Incorporation of the intercellular adhesion molecule ICAM-1 into human immunodeficiency virus type 1 (HIV-1) particles increased virus infectivity on peripheral blood mononuclear cells (PBMCs) by two- to sevenfold. The degree of ICAM-1-mediated enhancement was greater for viruses bearing envelope glycoproteins derived from primary HIV-1 isolates than for those bearing envelope glycoproteins from laboratory-adapted strains. Treatment of target PBMCs with an antibody against LFA-1, a principal ICAM-1 receptor, was able to nullify the ICAM-1-mediated enhancement. The incorporation of ICAM-1 rendered HIV-1 virions less susceptible to neutralization by a monoclonal antibody directed against the viral envelope glycoproteins. Surprisingly, an antibody against ICAM-1 completely neutralized infection by ICAM-1-containing viruses, reducing the efficiency of virus entry by almost 100-fold. Thus, HIV-1 neutralization by an ICAM-1-directed antibody involves more than an inhibition of the contribution of ICAM-1 to virus entry.

Location of cis-acting regulatory sequences in the human T-cell leukemia virus type I long terminal repeat.

The location of cis-acting regulatory regions within the long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-1) was determined. The sequences present between nucleotides -350 and -55 (cap site +1) contain an enhancer element that is active in lymphoid and nonlymphoid cell lines. The sequences located near the "TATA" and RNA initiation sites contain a promoter, the activity of which can be augmented by homologous and heterologous enhancer elements. A region responsive to trans-acting transcription factors present in HTLV-I- and HTLV type II-infected cells is located between nucleotides -159 and +315. HTLV-I LTR deletion mutants respond in a similar manner both to the trans-acting factors present in infected cells and to the tat protein encoded by the x-lor region of the genome, thus providing further evidence that the tat protein mediates transcriptional trans-activation of the LTR in HTLV-infected cells.

The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat.

The location of cis-acting regulatory sequences within the long terminal repeat (LTR) of the human T cell lymphotropic virus type III (HTLV-III/LAV) was determined. An enhancer element capable of increasing the rate of transcription from a heterologous promoter, irrespective of distance and orientation, is located between nucleotides -137 and -17 (cap site +1). The promoter sequences present near the TATA box respond to heterologous enhancers. The sequences present between nucleotides -17 and +80 are responsive to HTLV-III-associated trans-acting regulatory factors. Activation of these sequences by the viral regulatory factors requires the presence of a functional enhancer. The enhancer requirement is nonspecific, as the enhancer sequences of RSV, HTLV-I, and SV40 can functionally replace the HTLV-III enhancer. These findings define a new type of regulatory element, provide insight into the mechanisms that regulate HTLV-III gene expression, and may help to explain the effects of this virus on infected cells.

Transforming potential of a human protooncogene (c-fps/fes) locus.

The protooncogene c-fps/fes is a single vertebrate locus homologous to both the fps transforming gene of Fujinami avian sarcoma virus and the fes transforming gene of two feline sarcoma virus isolates. The human c-fps/fes locus has been previously cloned and characterized. We report that a recombinant gene in which 3' human c-fps/fes sequences replace over 80% of the feline sarcoma virus fes sequences transforms NIH 3T3 mouse fibroblasts and encodes a protein kinase. Cells transformed by this recombinant possess increased phosphotyrosine levels. These observations demonstrate that a large carboxyl portion of a human c-fps/fes protein product can functionally complement a retroviral transforming protein.