1692 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 6. ATPase domain of 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. Cells were labeled with [35S]-cysteine/methionine for 1 h for IP with Gag and b-Actin antibody. (A) Evaluation of rescue by siRNA-resistant FLAG-RHA by IP (top panel). Immunoblot of total cell protein with indicated antiserum veriﬁed RHA down-regulation, expression of siRNA-resistant FLAG-RHA and equal protein loading, respectively (bottom panel). (B) Evaluation of rescue by siRNA-resistant FLAG-RHA K417R by IP (top panel). Immunoblot of total cell protein with indicated antiserum veriﬁed RHA down-regulation, expression of siRNA-resistant FLAG-RHA K417R and equal protein loading, respectively (bottom panel). RHA is not necessary for packaging of virion RNA or LysRS Next, we examined virions produced from cells treated with RHA siRNA or non-silencing control siRNA. As described in the last section, RHA was down-regulated, veriﬁed by immunoblot, HIV-1NL4–3 was transfected with a second treatment of siRNA and after 48 h, progeny virions were harvested by centrifugation and virion- associated RNA was quantiﬁed by RT-real-time PCR. Immunoblotting veriﬁed down-regulation of RHA but no change in Actin or cell-associated Lys-tRNA synthetase (LysRS) (Figure 7A). However, HIV-1 Gag p55 and the processed forms of the Gag precursor protein p37 and p24 were reduced by RHA down-regulation. High exposure of the Gag immunoblot revealed that Gag-Pol polyprotein p160 is also reduced (right panel). As expected RT immunoblot detected similar reduction in RT p66, which is proteolytic pro- cessed from Gag-Pol p160. Vif, Rev and Nef immunoblot revealed concordant reduction in these virus proteins, sug- gesting that RHA is important for translation of genome-length, single-spliced and double-spliced mRNA. Next, equivalent quantities of virions were subjected to immunoblot (Figure 7B) and extraction of virion- associated RNA (5 ng Gag for immunoblot and 25 ng Gag for RNA extraction as measured by Gag ELISA). Gag immunoblot veriﬁed equivalent virion loading (Figure 7B, bottom panel). As expected, virions produced from cells treated with RHA siRNA are deﬁ- cient in RHA (Figure 7B, top panel). However they are not deﬁcient in cellular cofactor LysRS (23) nor processed RT (Figure 7B, middle panels). RT-real-time PCR with