Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3 splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3 end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3 end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3 end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.

Expression of the integrated HIV-1 provirus is achieved by overcoming multiple barriers to the processing, transport and utilization of the viral RNA. Some of the strategies involve viral encoded proteins (i.e. Rev, Gag). However, in large part it is host factors that play essential roles in the movement of HIV-1 RNA from the site of transcription to its ultimate encapsidation into new virions. Identifying these factors and their mechanism of action provides not only important insights into HIV-1 molecular biology but also that of the cell machinery itself. In this review, we highlight the viral and host factors regulating the splicing, polyadenylation, transport, and translation of HIV-1 RNA. The observations made underline the multiple fate decisions that must be made at each stage of the viral RNA metabolic pathway and highlight potential new avenues for controlling HIV-1 replication.

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Control of HIV-1 RNA processing is central to the replication of the virus. Previously, we demonstrated that the cellular protein Sam68 enhances HIV-1 structural protein expression and RNA 3' end processing. In this report, we show that Sam68 interacts with unspliced HIV-1 RNA and that other members of the STAR/GSG protein family also promote viral RNA 3' end processing. We define a portion of the GSG domain (Sam 97-255) as sufficient for enhancement of Rev-dependent expression. In contrast to Sam68, Sam 97-255 increases unspliced RNA processing only in the presence of Rev in 293T cells. In a different cell line, Sam 97-255 enhances HIV-1 gene expression without enhancing RNA 3' end processing, suggesting that stimulation of 3' end processing is not required for enhancement of HIV-1 gene expression. Overall, these results indicate that Sam68 and the mutants described affect the composition of the viral RNP to enhance viral protein synthesis.