Figure 4. RHA is necessary for eﬃcient translation of HIV-1 gag RNA. HEK 293 cells were transfected consecutively with non-silencing (Sc) or RHA siRNAs (RHA) and HIV-1NL4–3 for 48 h, labeled for 1 h with [35S]-cysteine/methionine and immunoprecipitation was performed in triplicate. Graph summarizes densitometry of [35S]-labeled Gag and b-Actin protein. Asterisks indicate statistically signiﬁcant diﬀerence from Sc siRNA control (P = 0.002). Immunoblotting veriﬁed RHA down-regulation (Figure 5A), and antisera to a-Tubulin and Histone H1 veriﬁed eﬀective nucleus and cytoplasm fractionation (Figure 5B). The gag and b-actin RNA levels were neither reduced by RHA down-regulation nor by the cytoplasmic accumula- tion of gag RNA (Figure 5C). Similar to the results of Figure 4, Gag IP on these samples determined that RHA down-regulation reduced de novo Gag protein syn- thesis but did not aﬀect Actin synthesis (data not shown). Given the similar levels of HIV-1 gag mRNA, the results Nucleic Acids Research, 2010, Vol. 38, No. 5 1691 Figure 5. RHA down-regulation does not aﬀect cytoplasmic accumu- lation of HIV-1 gag RNA. HEK 293 cells were transfected consecutively with non-silencing (Sc) or RHA siRNAs (RHA) and HIV-1NL4–3 for 48 h and aliquots harvested for fractionation or 1 h labeling with [35S]-cysteine/methionine. (A) Immunoblot of total cell protein with antiserum to RHA or b-Actin. (B) Immunoblot of nuclear or cytoplasmic extracts with Histone H1 or Tubulin antibody. (C) RT real-time PCR measured nuclear or cytoplasmic gag RNA and b-actin RNA. Cytoplasmic accumulation of gag RNA relative to b-actin is expressed as percentage. Translational eﬃciency is expressed as ratio of [35S]-labeled Gag protein to cytoplasmic gag RNA. indicate that RHA down-regulation reduces the translational eﬃciency of HIV-1 gag RNA. As summarized in Figure 5C, gag translation eﬃciency was reduced by a factor of 4. Next, we evaluated whether exogenous expression of siRNA-resistant FLAG-RHA is suﬃcient to rescue Gag protein synthesis. HEK 293 cells were treated with siRNAs and immunoblot determined that RHA was down-regulated (data not shown). These cells were transfected for 48h with siRNA-resistant FL-RHA and HIV-1NL4–3 and second treatment of siRNA, and then subjected to IP. RHA immunoblot veriﬁed RHA down-regulation and also detected the FL-RHA (Figure 6A, bottom). FLAG immunoblot veriﬁed expres- sion of siRNA-resistant FL-RHA and Actin immunoblot controlled for protein loading (Figure 6A, bottom). Similar to results of Figure 4, Gag IP indicated that RHA down-regulation severely impaired de novo Gag protein synthesis (Figure 6A, top). Furthermore, expres- sion of the siRNA-resistant FL-RHA rescued Gag protein synthesis (Figure 6A, top). Actin protein synthesis was not aﬀected by RHA down-regulation or FL-RHA expres- sion, consistent with previous results (2). To examine the role for the ATP-dependent helicase (22), we evaluated rescue by siRNA-resistant FL-RHA bearing K417R point mutation previously shown to eliminate ATP hydro- lysis (11,22). FLAG immunoblot veriﬁed expression of K417R (Figure 6B, bottom). Gag IP determined that the K417R mutant did not rescue Gag protein synthesis, suggesting that the ATP-dependent helicase activity is necessary for eﬃcient translation of HIV-1 gag (Figure 6B, top). As expected, Actin synthesis was not aﬀected by expression of K417R. The results demonstrated that RHA is necessary for eﬃcient transla- tion of gag RNA and the ATP-dependent helicase domain is involved.