AHIV Tat Protein and Tar Rna

Unlike simpler retroviruses that rely exclusively on host cellular machinery for gene expression, HIV, a lentivirus that causes the acquired immunodeficiency syndrome (AIDS), encodes additional regulatory proteins that work together with cellular machineries to further control viral replication (Barboric and Peterlin, 2005). Among these regulatory proteins, Tat, a small polypeptide of 101 amino acids in most clinical HIV-1 isolates, is recognized as a key factor essential for activating transcriptional elongation from the HIV LTR (long terminal repeat) and for productive viral replication (Brigati et al., 2003; Karn, 1999). Because the proteins that regulate elongation for many cellular and viral genes in a gene-specific fashion have not been identified, HIV Tat has been used as a model system to study this process. As a result, our understanding of not only the regulation of HIIV gene expression but also the general mechanism controlling eukaryotic elongation has benefited tremendously from the analysis of Tat and its host cellular cofactor P-TEFb.

In the absence of Tat, the HIV LTR generates short or so-called nonprocessive transcripts. The presence of Tat, however, results in a large increase in the level of transcripts that extend through the more than 9-kb HIV genome. Tat stimulation of the efficiency of transcriptional elongation is primarily responsible for this dramatic increase in the level of full-length HIV transcripts (Barboric and Peterlin, 2005). Tat stimulates elongation by recognizing the trans-acting-response (TAR) RNA element. Located at the 5' end of the nascent viral transcript (nucleotides +1 to +59), TAR forms a stem-loop structure (Fig. 14.4). The specific binding of Tat to TAR is primarily dependent on the 3-nt bulge and immediately flanking sequences in the double-stranded RNA region just below the apical loop. This binding enables the recruitment of Tat and through Tat the key cellular cofactor P-TEFb to the paused Pol II on the viral promoter (Barboric and Peterlin, 2005; Jones, 1997; Karn, 1999).

B:P-TEFb Stimulation of HIV Transcriptional Elongation through Tat and TAR

Besides functioning as a general elongation factor capable of overcoming the pausing of Pol II during early elongation (Price, 2000), P-TEFb was also identified as a Tat-associated kinase (so-called TAK) specifically required for HIV transcription and viral replication (Herrmann and Rice, 1995; Yang et ah, 1997). RNA Pol II transcribing the integrated HIV proviral DNA has a particularly strong tendency to pause and then terminate close to the start site, producing only short transcripts. Preventing Pol II from stalling is essential for HIV transcription, during which P-TEFb is recruited to the nascent mRNA by Tat (Garriga and Grana, 2004; Jones, 1997; Price, 2000). Unlike most DNA sequence-specific transcription activators, Tat stimulates polymerase elongation by recognizing the HIV TAR RNA structure and recruiting P-TEFb to the HIV promoter through the Tat:CycTl interaction and the formation of a ternary complex containing P-TEFb, Tat and TAR (Garber et al., 1998; Wei et al., 1998; Zhang et al., 2000). Once recruited, P-TEFb phosphorylates the Pol II CTD and stimulates transcriptional elongation to produce the full-length HIV-1 transcripts (Fig. 14.4).

A definitive proof that P-TEFb is the specific cellular cofactor for Tat function came from studies of HIV transcription in rodent cells. Tat activity has been shown to be species-specific, as it trans-activates the HIV LTR efficiently in many human and primate cell types but not in cells of other species (e.g., yeast, Drosophila, and rodent cells) (Jones and Peterlin, 1994), suggesting that there exists a species-restricted cellular cofactor for Tat function (Alonso et al., 1994; Hart et al., 1993). To investigate whether human P-TEFb contributes

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