Figure 4
From: The role of A-kinase anchoring protein 95-like protein in annealing of tRNALys3to HIV-1 RNA
Decreased cellular HAP95 results in reduced annealing of tRNALys3to viral RNA. (A) Total viral RNA was isolated from purified HIV-1 particles, and tRNALys3 annealed to viral RNA in the cell was extended using reverse transcriptase by either 6 nucleotides (+6 nt) or 1 nt (+1 nt). Upper panel: The extended tRNALys3 products are resolved by 1D PAGE. The control gel shows that equal amounts of viral RNA were used in each extension reaction. Lower panel: The relative amounts of +6 or +1 nt extended tRNALys3 products are represented graphically. Shown are the mean values ± SD of 3 experiments. *, P < 0.05. (B) Rescue of the annealing of tRNALys3 inhibited by siRNAHAP by expression of HAP95 mRNA resistant to siRNAHAP. Upper panel: Western blots of cellular lysates probed with anti-HAP95, anti-Flag, or anti-β-actin. Lower panel: Western blots of viral lysates containing equal amounts of CAp24. (C) +6 nt extension assay. Upper panel: +6 nt-extended tRNALys3 detected in 1D PAGE. Lower panel: The relative amounts of +6 nt extended tRNALys3 products are represented graphically. *, P < 0.05 compared to the results obtained with virions produced from the cells expressing Flag tag only. (D) Quantitation of tRNALys3 in HIV-1 particles by real-time RT-PCR. Shown are the averages of fold change ± SD of 3 experiments. (E) Viral infectivity assay was performed as described in Figure 2 legend. *, P < 0.05 compared to the results obtained with virions produced from the cells expressing Flag tag only. (F) HIV-1 cDNA synthesis in the newly BH10-infected SupT1 cells. The amounts of viral RNA or newly synthesized cDNA in the cells were quantitated by real-time RT-PCR or real-time PCR. *, P < 0.05 compared to the results obtained with virions produced from the cells expressing Flag tag only.