genome-wide studies (8). Previous RHA overexpression studies detected increased HIV-1 gene expression as measured by HIV-1 LTR-luciferase reporter gene activity (9); an eﬀect on balanced expression of the unspliced HIV-1 gag RNA; and increased virion protein production (10). The results suggested RHA aﬀects HIV-1 transcription and/or post-transcriptional expression. Biochemical analysis has revealed that RHA can act as a scaﬀold to bridge the association of CREB-binding protein and RNA polymerase II (11). And a role for inter- action with HIV-1 RNA was invoked from Northwestern analysis detecting RHA interaction with the HIV-1 trans- activation response element (TAR) within the R region of the HIV-1 50 LTR (9); TAR is the target for the essential HIV-1 Tat transcriptional trans-activator. Recently, Liang and colleagues determined that RHA interacts with HIV-1 Gag and is packaged into HIV-1 particles in an RNA-dependent manner. RHA down-regulation by siRNA decreased HIV-1 infectivity that was attributed in part to lower reverse transcriptase (RT) activity (12). In summary, RHA appears to inﬂuence HIV-1 gene expression and possibly the process of virus assembly. A possible role for RHA in translation of the virus and possible involvement of the ATP-dependent helicase activity remains an open issue. Given the identiﬁcation of RHA-dependent PCE activity in the RU5 of several animal retroviruses, we evaluated the RU5 regions of human retroviruses, HTLV-1 and HIV-1, for PCE reporter activity. We also examined the eﬀect of RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1 provirus, virion production, content and infectivity in PBMC. The results demonstrated that RU5 of HIV-1 and HTLV-1 confer orientation-dependent PCE reporter activity, and that RHA aﬀects two steps in replication of HIV-1: (i) translation of the viral RNA and (ii) infectivity of progeny virions. MATERIALS AND METHODS Cell culture, transfection, infection HEK 293 and HeLa TZM-bl (13–16) were cultured in Dulbecco’s modiﬁed eagle medium (Invitrogen), 10% FBS (Invitrogen) and 1% antibiotic (Gibco) at 37 C/5% CO2. Peripheral blood mononuclear cells (PBMCs) were isolated as described previously (17) and were stimulated by 5 mg/ml PHA and 100 U/ml recombinant human Interleukin-2 (Roche) for 2 days in RPMI 1640 medium (Invitrogen), 10% FBS, 1% antibiotic and then with supplementation of 50 U/ml Interleukin-2 at 37 C/5% CO2. Cultures of 2.5 105 HEK 293 cells per 35 mm dish were incubated overnight in 2 ml medium and then transfected with 2 mg indicated reporter plasmid, 150 ng pGL3 ﬁreﬂy luciferase plasmid in 2 HBS as described (3) for 2 days. Cells were harvested in PBS, resuspended in 50 ml NP40 lysis buﬀer and 5 ml lysate was tested for Luciferase activity (Promega) as described (18) or diluted for HIV-1 Gag p24 ELISA (Zeptometrix). Construction of PCE reporter plasmids is provided in Nucleic Acids Research, 2010, Vol. 38, No. 5 1687 Supplementary Data. HTLV-1 reporters contain the Tax-dependent U3 reporter and were co-transfected with 500 ng HTLV-1 tax-1 expression plasmid. Methods for RNA isolation and analysis are provided in Supplementary Data. For siRNA transfection, 1 106 HEK 293 cells per 10 cm dish were cultured overnight and treated with 50 ml Oligofectamine (Invitrogen) and 20 mM indicated siRNA (Dharmacon) in 2 ml OptiMEM media (Gibco) and 5 ml DMEM without serum. Previously described (2) RHA siRNA: RHA1 UAGAAUGGGUGGAGAAG AAUU and RHA2 GGCUAUAUCCAUCGAAAU UUU and non-silencing scrambled (Sc) siRNA UAGAC UAGCUGACGAGAAAUU were transfected for 4 h, and the cultures were supplemented with 3.5 ml DMEM containing 30% FBS, 1% antibiotic and incubated for 48 h. These cells were split, cultured overnight. After second siRNA treatment, cells were cotransfected with 10 mg pNL101 HIV-1NL4–3 and 5 mg siRNA-resistant FLAG-tagged RHA (FL-RHA) or 5 mg siRNA-resistant FL-RHA in 1:3 DNA to FuGene6 (Roche) ratio by man- ufacturer protocol. RHA down-regulation was veriﬁed by immunoblot as described in Supplementary Data and ref. (2). For infection experiments, HIV-1NL4–3 was propagated by transfection of cultures of 1 106 HEK 293 cells per 10 cm dish with 10 mg pNL101 in FuGene6 for 48 h. Virus-containing cell-free supernatant was used for Gag p24 ELISA, and 2 ng HIV-1 p24 was used to infect 1 106 PBMC or 2 105 HeLa TZM-bl cells (13–16). As described previously (19), virus growth on PBMC target cells was monitored by Gag ELISA on cell-free supernatant. HeLa TZM-bl cultures were harvested 48 h post-infection, extracted in the NP40 lysis buﬀer, 10 mg aliquots determined by Bradford assay, and used for Luciferase assay. Statistical analyses Four independent PCE reporter transfections were per- formed and data were log-transformed for the statistical analyses. ANOVA model was used to study the diﬀerences in Gag protein level and gag RNA level. Dunnett’s method was used to adjust for multiple comparisons. Data are pre- sented as mean fold change with corresponding P-values (Figures 1 and 2). A one sample t-test compared the fold change diﬀerences in HIV-1 Gag production between RHAi and Sci groups (Figure 4). Three replicates were obtained for the HIV-1 infection assays. Data were analyzed using an ANOVA model that included group, time and their interaction term. Holm’s method was used to adjust for multiplicity (Figures 3 and 8). RESULTS HTLV-1 RU5 provide orientation-dependent PCE activity Previous results demonstrated that the 50 LTR of HTLV-1ACH confers PCE activity in reporter assays (5). PCE reporter activity is deﬁned by Rev/Rev responsive element (RRE)-independent expression of intron- containing HIV-1 gag RNA (3–5). Site-directed