1690 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 2. HIV-1 RU5 is necessary for PCE activity and interaction with RHA. (A) Drawing of HIV-1 PCE-gag reporter plasmid with HIV-1 R depicted in green and U5 in orange with corresponding sequence shown below. Arrow indicates transcription start site; 50 ss, splice site; pA(n), polyadenylation signal. Dashed line denotes deletion of R and reverse arrow indicates antisense orientation (AS) of RU5. HEK 293 cells were transfected with the indicated plasmid, Gag production was measured by Gag p24 ELISA and gag mRNA was quantiﬁed by RT-real-time PCR. ANOVA model was used to study the diﬀerences in Gag protein level and gag RNA level. Data are presented as mean fold change with corre- sponding P-values. Results are presented as mean fold change relative to pHIV-1U5 with P-value indicated in parentheses. Asterisks indicate statistically signiﬁcant diﬀerence (P 0.05). (B) RNA aﬃnity chromatography was performed with HeLa nuclear extract and equal moles of indicated HIV-1 RNA or c-myc RNA bait. High aﬃnity proteins were eluted in 2 M KCl and immunoblotted with RHA antiserum. Lanes: input lysate; M, marker; Beads, reaction with no RNA control and indicated RNA bait. Figure 3. RHA is necessary for eﬃcient production of HIV-1 Gag. HEK 293 cells were transfected consecutively with non-silencing (Sc) or RHA siRNAs (RHA) and HIV-1NL4–3. (A) Immunoblot of total cell protein with antiserum to RHA or a-Tubulin veriﬁed RHA down-regulation. (B) Cell-associated (n = 5) and cell-free (n = 16) Gag levels measured by Gag immunoblot or Gag ELISA, respectively. Asterisks indicate statistically signiﬁcant diﬀerence from Sc siRNA (P = 0.0006 for cell-associated and P < 0.0001 for cell-free Gag, respectively). (C) Cell-free Gag production measured by Gag ELISA (n = 3). Asterisks indicate statistically signiﬁcant diﬀerence from Sc siRNA at indicated time point (P 0.0001).