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).     

Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps.

We have examined, using self-cleaving ribozymes, the intracellular trans-activation kinetics of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by viral protein Tat. Experiments were designed to effect a competition (during RNA chain elongation) between cleavage of a nascent RNA containing the Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-activation. We found that fast self-cleavage of nascent TAR-containing RNA abolished Tat trans-activation. Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate-limiting step in HIV-1 LTR trans-activation is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA polymerases that are tethered to functional TAR.     

TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein.     

Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity.

The Epstein-Barr virus latent membrane protein (LMP) is an integral membrane protein that is expressed in cells latently infected with the virus. LMP is believed to play an important role in Epstein-Barr virus transformation and has been shown to induce expression of several cellular proteins. We performed a series of experiments that demonstrated that LMP is an efficient transactivator of expression from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). Mutation or deletion of the NF-kappa B elements in the LTR abolished the transactivation, indicating that the LMP effect on HIV expression was due to induction of NF-kappa B activity. Experiments in which the HIV-1 Tat protein was coexpressed in cells together with LMP showed that Tat was able to potentiate the transactivation. Surprisingly, a synergistic effect of the two proteins was observed even in the absence of the recognized target region for Tat (TAR) in the HIV-1 LTR.     

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.     

Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function.

Expression of the human immunodeficiency virus type 1 (HIV-1) is strongly activated by Tat. The proper action of Tat requires three elements: TATAA, TAR, and upstream motifs in the HIV-1 long terminal repeat. We show here that the correct spatial arrangement among Tat, Sp1, and TATAA crucially influences HIV expression. Under conditions in which basal promoter activity is unperturbed, distancing Sp1 from TATAA markedly affected Tat trans activation. An increase in the Sp1-TATAA distance from 18 to 101 nucleotides (depending on the inserted sequence) rendered HIV-1 either partially or wholly replication defective. This critical dependence on spacing suggests that Tat-, Sp1-, and TATAA-binding factors must correctly contact each other for optimal expression and replication of HIV-1.