Nucleic Acids Research, 2010, Vol. 38, No. 5 1693 Figure 7. RHA down-regulation reduces virus protein but not HIV-1 RNA packaging. HEK 293 cells were transfected consecutively with scrambled (Sc) or RHA (RHA) siRNAs and HIV-1NL4–3 provirus and cellular protein and RNA were isolated. Gag p24 ELISA on cell-free medium was performed. (A) Immunoblot of total cell protein (10 mg) with antiserum to Gag, RHA, LysRS, Vif, Nef, Rev or b-Actin. (B) Immunoblot of virion preparation (equivalent to 5 ng of p24) with antiserum to RHA, LysRS, RT, Gag. (C) RT-real-time PCR deter- mined copy number. aGag copy number in virion preparation equiva- lent to 25 ng of p24. bCellular gag RNA copy number standardized to b-actin RNA. gag primers detected similar amounts of virion-associated RNA (Figure 7C). In this particular experiment, RT-real-time PCR on total cellular RNA detected a minor reduction in cell-associated gag RNA, Figure 7C. The results indicate that viral RNA packaging eﬃciency was not reduced by RHA down-regulation. In sum, analysis of HIV-1NL4–3 demonstrated that RHA down-regulation reduces the eﬃciency of HIV-1 RNA translation. However, virion associated LysRS and viral RNA are not reduced, suggesting that RHA is not neces- sary for their packaging into RHA-deﬁcient particles. RHA is important for HIV-1 infectivity on primary cells To examine the eﬀect of RHA down-regulation on HIV-1 infectivity, equivalent cell-free virion preparations were Figure 8. RHA down-regulation reduces infectivity of progeny HIV-1. (A) HEK 293 cells were transfected with scrambled (Sc) or RHA (RHA) siRNAs, and then second dose of siRNA and HIV-1NL4–3 for 48 h. Cell-free virus equivalent to 2 ng Gag was used to infect activated PBMCs. Virus growth on PBMC was measured by Gag ELISA (n = 3). Asterisks indicate statistically signiﬁcant diﬀerence from Sc siRNA control was observed at day 6, 9 and 12 (P 0.0005, 0.0008, 0.0174). (B) HEK 293 cells were transfected with Sc or RHA siRNAs, and then second dose of siRNA with either empty vector or siRNA-resistant FLAG-RHA and HIV-1NL4–3. Cell-free virus equivalent to 2 ng Gag was used to infect TZM-bl cells and luciferase activity determined at 48 h (n = 3). used for infection of human PBMCs or a HeLa-based Luciferase reporter cell line, TZM-bl integrated with HIV-1 LTRluc reporter gene (13–16). Gag ELISA was performed on the supernatant medium from producer cells treated with RHA siRNA or Sc siRNA, and infections were performed with 2 ng Gag aliquots of virions deﬁcient in RHA or containing RHA. Virus growth on PBMC in triplicate experiments determined that RHA-deﬁcient virions exhibit reduced Gag production in PBMC (Figure 8). Reduced virus growth was observed at days 6, 9 and 12, indicating virus replication is impaired (P < 0.0005, 0.0008, 0.0174, respectively, Figure 8A). Similarly, virus infectivity on TZM-bl cells was reduced and expression of siRNA-resistant RHA was suﬃcient to rescue HIV-1 infectivity (Figure 8B). The results demon- strate that RHA down-regulation impaired the infectivity of progeny virions. In conclusion, RHA plays two critical roles in the virus producer cell: (i) RHA is necessary for eﬃcient HIV-1 translation; (ii) RHA assembled into HIV-1 virions promotes infectivity on primary human cells.