Rev/RRE-independent expression of HIV-1 gag RNA. Gag levels increased 2-fold in response to RU5 in the sense but not the antisense orientation (pHTLV-1RU5, P = 0.03 and pHTLV-1AS, P = 0.22). HTLV-1 R in either the sense or antisense orientation did not increase Gag production. HTLV-1 U5 was inhibitory to Gag protein production in either sense (P = 0.01) or antisense orientation (P 0.01) orientation, which may be attribut- able to RNA structural features that impede ribosome scanning (20,21). To examine the abundance of steady-state gag RNA, polyA+ RNA was prepared from these samples; reverse transcribed and subjected to real-time PCR with gag and -actin primers. Signiﬁcant diﬀerences in gag mRNA levels were not observed among the samples, indicating the increase in Gag production is not attributable to increased mRNA accumulation (Figure 1A). The results indicated that HTLV-1 R or U5 are not suﬃcient for PCE activity and the combination of R and U5 function in an orientation-dependent manner to provide HTLV-1 PCE reporter activity. Association with RHA was evaluated by RNA aﬃnity chromatography and immunoblot by the protocol used to identify speciﬁc interaction of RHA with SNV PCE (2). Biotinylated HTLV-1 RU5, R and U5 transcripts or c-myc control RNA were synthesized with T7 RNA polymerase and puriﬁed on G-25 columns. Equimolar amounts (15 mM) of each transcript were bound to streptavidin agarose beads, incubated with HeLa nuclear lysate, and washed with binding buﬀer. Low aﬃnity RNA-binding proteins were eluted by progressive treat- ment with elution buﬀer with 40, 100 and 200 mM KCl. High aﬃnity RNA-binding proteins were eluted in 2 M KCl and immunoblotted with antisera to RHA (Figure 1B). RHA co-precipitated with HTLV-1 RU5. RHA was less abundant in reactions with equimolar amounts of R or U5, which correlated with the dearth of PCE activity. RHA was not enriched on beads alone, antisense RU5 (AS-RU5) or c-myc 50 UTR RNA, indicating that RHA interaction with RU5 was not attrib- utable to non-speciﬁc interaction. HIV-1 RU5 confer PCE activity A similar approach was used to examine HIV-1 RU5 for PCE activity. As summarized in Figure 2A, PCE reporter plasmids that encode the SNV U3 promoter and HIV-1 leader and gag open reading frame were evaluated in four independent transfections. Baseline Gag production was observed from pHIV-1U5 that lacks R and pHIV-1RU5AS that contains RU5 in the antisense orien- tation did not change Gag production (P = 1). The pHIV-1RU5 that contains RU5 in the sense orientation exhibited Rev/RRE-independent Gag production at a level 3.5-fold greater than baseline (pHIV-1RU5, P = 0.005). A similar level of PCE activity is observed from SNV PCE reporter pYW100 that contains SNV RU5 (4.9-fold). RT-real-time PCR determined steady- state gag RNA levels were similar among the samples (Figure 2A), indicating the increase in Gag production is not attributable to increased accumulation of the mRNA. Nucleic Acids Research, 2010, Vol. 38, No. 5 1689 RNA aﬃnity chromatography and RHA immunoblot surveyed association of HIV-1 RU5 with RHA. RHA co-precipitated with HIV-1 RU5 but was less abundant in equimolar HIV-1 R or U5 (Figure 2B). RHA was not enriched in reactions with beads alone or c-myc RNA, indicating that RHA interaction with RU5 was not attrib- utable to non-speciﬁc interaction. To examine physical interaction in cells, in addition, RNA co-IP assays were performed on cells co-transfected with pHIV-1 RU5 and pCMV-FL-RHA or empty pFL plasmid. Cytoplasm was harvested in aliquots for IP with FLAG antibody fol- lowed by RNA isolation from the immunoprecipates, or isolation of cytoplasmic RNA. RNA from the immunoprecipates was treated with random hexamers in reactions with RT and without RT, and reactions with primers speciﬁc for gag or junD control were subjected to real-time PCR (2). The copy number of gag in the cytoplasmic RNA was similar between samples with FL-RHA or empty FL (2.7 105 and 3.9 105, respec- tively). The gag copy number in FL-RHA co-precipitate (3.5 104) was enriched >3-fold over the empty FL control, which provided the detection limit of the assay (1.0 104). In sum, the RNA co-precipitation results agree with the RNA aﬃnity chromatography and further dem- onstrate that gag RNA interacts with RHA in vivo. We conclude that HIV-1 U5 is not suﬃcient for PCE activity and RU5 function in an orientation-dependent manner to provide PCE reporter activity. RHA modulates the cytoplasmic utilization of HIV-1 gag RNA To evaluate whether RHA is necessary for eﬃcient trans- lation of HIV-1, RHA was down-regulated with two previously characterized siRNAs or non-silencing scrambled control siRNA (Sc) for 48 h (2) (data not shown). These cells were transfected with HIV-1NL4–3 and the same siRNA for 48 h and immunoblot veriﬁed RHA down-regulation (Figure 3A). Total cellular protein was subjected to Gag immunoblot and cell-free supernatant medium was subjected to Gag ELISA. The results indicate signiﬁcant reductions in cell-associated (P = 0.0006) and cell-free Gag (P < 0.0001; Figure 3B). The equivalent reductions in cell-associated and cell-free Gag indicated that RHA down-regulation aﬀects the rate of Gag expression, but does not impair particle assembly or release. ELISAs were also performed on cell-free supernatant collected sequentially over 48 h. These results also indicated that RHA down-regulation delayed the onset of virion production by twelve hours and reduced absolute Gag production (Figure 3C). Metabolic labeling measured the eﬀect of RHA down-regulation on Gag protein synthesis. After 1 h incu- bation with [35S]-cysteine/methionine, IP was performed with Gag or b-Actin antibody. As shown in Figure 4, RHA down-regulation signiﬁcantly reduced de novo Gag protein synthesis, but did not reduce b-Actin protein syn- thesis (P = 0.002). To quantify the eﬀect of RHA down-regulation on steady-state gag RNA abundance and cytoplasmic accumulation, nuclear and cytoplasmic RNA were isolated and subjected to RT-real-time PCR.