Figure 3

Identification of an antisense RNA from HIV-1-infected cells. (A) A schematic representation of the HIV-1 asRNAs and primers for 3' RACE. The black arrow represents the ASP-L transcript characterized in this study, and dotted arrows are the ASP mRNAs as previously described [8, 14]. The bold green arrow indicates the position of the potential ORF, ASP. (B and C) Results of 3' RACE. ‘MAGIC5A-HIV’ stands for HIV-1 infected MAGIC-5A and ‘293 T-asHIV’ for HEK293T cells transfected with pME18S-asHIV. Lane 1 and 5, no infection control or mock transfected cells; Lane 2 and 6, cells with HIV-1 infection or transfected with pME18S-asHIV; Lane 3 and 7, no RTase control; Lane 4 and 8, no RT-primer control; M, 100 bp marker. (B) The result of 3′ RACE PCR using the p4R primer (upper panel) and the p3R primer (lower panel). The asterisk indicates the PCR product representing ASP-L. ‘II’, ‘III-iii’ and ‘IV-ii’ represent the PCR product derived from transcripts II, III-iii and IV-ii, respectively, described in Figure 1. (C) Upper panel: Results of semi-nested PCR with the YT001 primer. The white asterisks represent ASP-L-variant forms; the upper asterisk indicates ASP-L2, the lower one ASP-L1. The yellow asterisks represent transcript III-iii. Results of the sequencing analyses are shown in the bottom panel, where arrows and upper case letters indicate the termination sites. The bold arrow, end of the major transcript; the blue upper letters, a putative polyadenylation signal predicted by HCpolya (see Methods); the underline, putative polyadenylation elements of ASP-L. (D and E) Identification of transcription start site (TSS) of ASP-L. (D) Results of antisense-specific RT-PCR with two kinds of reverse primers. Locations of p5Ftag (Tag-RT-primer), 9418r, and 9538r are described in E. Lanes 1–6 are the same as in Figure 2B. (E) A schematic description of ASP-L RNA. The upper case letter C in the sequence indicates the position of ASP-L TSS.