Effects of Sam68 on HIV-1 RNA Processing and Gene Expression

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.Ph