Human chromosome 12 encodes a species-specific factor which increases human immunodeficiency virus type 1 tat-mediated trans activation in rodent cells.

The human immunodeficiency virus type 1 (HIV-1) tat protein functions at a much lower level in rodent cells than in human cells. This species-specific difference in trans activation appears to be due to the lack of a functional homolog of a human cofactor for tat in rodent cells. Using HIV-1 long terminal repeat-driven human growth hormone as a reporter plasmid, we found that the tat-mediated trans activation functions at a level 5- to 20-fold lower in rodent cells than in human cells. Stable rodent-human hybrid cells containing only human chromosome 12 support a dramatically higher degree of trans activation. Thus, human chromosome 12 encodes a species-specific HIV-1 tat cofactor which, at least partially, restores high levels of tat-mediated trans activation. Chromosome 6 also appears to provide an additional factor which enhances HIV-1 tat-mediated trans activation in murine cells.     

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.     

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells.

Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).     

Human immunodeficiency virus type 1 Tat-mediated trans activation correlates with the phosphorylation state of a cellular TAR RNA stem-binding factor.

Protein kinase C (PKC) is involved in the mitogenic stimulation of cell proliferation and has recently been reported to be essential for Tat-mediated trans activation. We have determined that RNA binding of a cellular factor which specifically interacts with the trans-activation response region (TAR) is blocked in cells depleted of PKC activity by chronic phorbol myristate acetate stimulation. We also show that nuclear extracts can be depleted of the cellular TAR-binding factor by in vitro treatment with purified protein phosphatase 2A. Furthermore, TAR RNA-binding activity can be partially restored to depleted nuclear extracts in vitro by addition of PKC. Chimeric constructs in which the Tat protein is artificially tethered to viral RNA show PKC independence for Tat-mediated trans activation. Specific mutations in the TAR RNA stem region which cause reduced binding of host cell factor in vitro also cause reduced Tat-mediated trans activation in vivo. Together, these results suggest that phosphorylation-dependent binding of a cellular cofactor to TAR RNA is an essential step in Tat-mediated trans activation. Deciphering the regulation of Tat-mediated trans activation by phosphorylation will be critical in fully understanding the regulation of human immunodeficiency virus type 1 activation.     

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.     

Complement activation by human monoclonal antibodies to human immunodeficiency virus.

It has been shown that the incubation of human immunodeficiency virus (HIV) with polyclonal antibodies from HIV-infected persons and complement results in complement-mediated neutralization due, at least in part, to virolysis. The current study was performed to determine whether any of a panel of 16 human monoclonal antibodies to HIV could activate complement and, if so, which determinants of the HIV envelope could serve as targets for antibody-dependent complement-mediated effects. Human monoclonal antibodies directed to the third variable region (V3 region) of HIVMN gp120 induced C3 deposition on infected cells and virolysis of free virus. Antibodies to two other sites on HIVMN gp120 and two sites on gp41 induced few or no complement-mediated effects. Similarly, only anti-V3 antibodies efficiently caused complement-mediated effects on the HIVIIIB isolate. In general, the level of C3 deposition on infected cells paralleled the relative level of bound monoclonal antibodies. As expected, pooled polyclonal antibodies from infected persons were much more efficient than monoclonal antibodies inducing C3 deposition per unit of bound immunoglobulin. Treatment of virus or infected cells with soluble CD4 resulted in increases in anti-gp41 antibody-mediated virolysis and C3 deposition but decreases in anti-V3 antibody-mediated virolysis and C3 deposition. In general, virolysis of HIV was more sensitive as an indicator of complement-mediated effects than infected-cell surface C3 deposition, suggesting the absence of or reduced expression of functional complement control proteins on the surface of free virus. Thus, this study shows that human monoclonal antibodies to the V3 region of gp120 are most efficient in causing virolysis of free virus and C3 deposition on infected cells. Elution of gp120 with soluble CD4 exposes epitopes on gp41 that can also bind antibody, resulting in virolysis and C3 deposition. These findings establish a serologically defined model system for the further study of the interaction of complement and HIV.     

Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs.

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.     

Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR.

trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA. Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined. Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo. Rather, the activation domain is also required and must be juxtaposed to the basic domain. Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.     

Functional comparison of the basic domains of the Tat proteins of human immunodeficiency virus types 1 and 2 in trans activation.

The trans-activator Tat proteins coded by human immunodeficiency virus type 1 (HIV-1) and HIV-2 appear to be similar in structure and function. However, the Tat protein of HIV-2 (Tat2) activates the HIV-1 long terminal repeat (LTR) less efficiently than Tat1 (M. Emerman, M. Guyader, L. Montagnier, D. Baltimore, and M. A. Muesing, EMBO J. 6:3755-3760, 1987). To determine the functional domain of Tat2 which contributes to this incomplete reciprocity, we have carried out domain substitution between Tat1 and Tat2 by exchanging the basic domains involved in Tat interaction with its target trans-activation-response (TAR) RNA structure. Our results indicate that Tat1 proteins containing substitutions of either 8 or 14 amino acids of the basic domain of Tat2 exhibited reduced trans activation of the HIV-1 LTR by about 1/20 or one-fourth the level induced by wt Tat1. In contrast, Tat2 containing a substitution of the 9-amino-acid basic domain of Tat1 trans activated HIV-1 LTR like native Tat1. A substitution of the highly conserved core domain of Tat2 with that of Tat1 did not have any significant effect on trans activation of the HIV-1 LTR. These results indicate that the basic domain of Tat2 contributes to its inefficient trans activation of the HIV-1 LTR. Mutation of an acidic residue (Glu) located between the core domain and the Arg-rich basic domain of Tat2 at position 77 to a Gly residue increased the activity of Tat2 substantially. These results further suggest that the presence of an acidic residue (Glu) adjacent to Arg-rich sequences may at least partially contribute to the reduced activity of the Tat2 basic domain.     

UV activation of human immunodeficiency virus gene expression in transgenic mice.

Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck.