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. 2006 Oct 3;4(10):e332. doi: 10.1371/journal.pbio.0040332

Figure 9. MHIV-1 gag Mutant Does Not Incorporate tRNA Species with RTC Nuclear Import Activity.

Figure 9

(A) Viral RNA (1.5 μg) was extracted from purified HIV-1 (lane 1) or MHIV-1 gag mutant (lane 2) and separated onto a long (50 cm) 15 % denaturing PAGE followed by SYBR Gold staining. There was a 10-bp DNA ladder (lane St), 25-ng tRNALys1,2 size G2 RNA + C tail (see Table S2) (lane 3), 25-ng tRNALys1,2 size G2 RNA + CC tail (lane 4), and 25-ng tRNALys1,2 size G2 RNA + CCA tail (lane 5). Asterisks indicate the three small RNA bands found in HIV-1; arrows indicate viral-specific small RNA molecule common to both viruses.

(B) Single-cycle infection assays in cell cycle–arrested cells. Cells were treated with aphidicolin for 24 h to induce G1/S arrest, infected with the same dose of wild-type HIV-1 or MHIV gag mutant, and analyzed for GFP expression by flow cytometry 24 h after infection. Bars represent the average value ± standard deviation of two experiments.

(C) Nuclear import of YOYO-1–labelled HIV-1 RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and buffer (ctr–) or HIV-1 small RNAs (HIV-1 sRNA) or MHIV-1 gag mutant small RNAs (MLV/HIV-1 sRNA) (30ng + 70-ng carrier siRNA) after elution from the gel. Nuclear import of the eluted small viral RNAs from wild-type HIV-1 and MHIV gag mutant in the absence RTCs was performed as an additional negative control (HIV-1 sRNA and MLV/HIV sRNA, respectively).