Figure 5

Domains of vmr11 RNA required for PTEN or TRN-2 inhibition. (A) vmr11mutants (upper panel): Base substitutions are shown in red. Western blots of PTEN and TRN-2 proteins (shown in lower panel): PPH cultures were transfected (twice, at 0 and 24 hours) with 50 nM vmr11 ‘mimic’ or one of the seven vmr11 mutants as indicated; total cell protein (for TRN-2), or the nuclear protein (for PTEN) was analyzed by Western blots. TRN-2 or PTEN protein levels were determined from two independent experiments, shown below each lane. β-actin or Lamin B1 was used as loading controls for TRN-2 and PTEN, respectively. (B) Virus production is positively correlated with loss of nuclear PTEN: 10⁵ PPH cells each were transfected with 1 μg HCV genomic RNA. A day later the cultures were either mock-transfected or transfected with vmr11 ‘mimic’ or with one of the seven vmr11 mutants (indicated in panel A). The oligonucleotide transfections were repeated twice (at 24 and 48 hour). Virus released in the culture media was harvested at 24-hour intervals as indicated. Viral RNA was analyzed by qRT-PCR. All viral RNA levels shown were normalized to the control, ‘mock transfected’ cells at 24 hour. (C) Loss of Nuclear PTEN and TRN-2 protein levels in cells transfected with vmr11 or vmr11 mutants: PTEN and TRN-2 proteins from the same cultures where virus released into the culture medium was quantitated (panel B), were analyzed by Western blots. Relative values of nuclear PTEN or TRN-2 proteins are shown below each lane.