1688 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 1. HTLV-1 RU5 is necessary for PCE activity and interaction with RHA. (A) Drawing of HTLV-1 PCE-gag reporter plasmid with HTLV-1 R depicted in blue and U5 in purple with corresponding sequence shown below. Arrow indicates transcription start site; 50 ss, splice site; pA(n), polyadenylation signal. Dashed line denotes deletion of RU5 and reverse arrow indicates antisense orientation (AS) of RU5. HEK 293 cells were transfected with indicated reporter plasmid and HTLV-1 tax-1 expression plasmid to trans-activate the HTLV-1 U3 promoter for 48 h. Gag protein production was measured by Gag p24 ELISA and gag mRNA was quantiﬁed by RT-real-time PCR in four independent experiments. ANOVA model was used to study the diﬀerences in Gag protein level and gag RNA level and data are presented as mean fold change with corresponding P-values relative to pHTLV-1 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 equimolar HTLV-1 RU5, RU5 AS, R, U5 or c-myc RNA bait. After sequential washes with KCl, 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. mutagenesis of SNV RU5 have determined that two func- tionally redundant stem-loop structures encoded by RU5 provide orientation-dependent PCE activity (5,6). To determine whether the R and U5 regions of HTLV-1 are necessary or suﬃcient for PCE activity, PCE reporter plasmids were generated (Supplementary Data) that contain the HTLV-1 U3 promoter and lack or contain R (nucleotides 1–220, relative to the transcription start site) and U5 (nucleotides 237–402) in the sense and antisense orientations (Figure 1A). The plasmids were cotransfected with tax-1 transcriptional trans-activator of the HTLV-1 U3 promoter. HIV-1 Gag levels were measured by ELISA. Results of four independent transfection experiments were evaluated for statistical trends (Figure 1A). Compared to pHTLV-1 that lacks RU5, pHTLV-1RU5 exhibited low but detectable