Figure 3
Silencing endogenous MOV10 has no significant effect on the production of infectious retrovirus particles for a panel of exogenous retroviruses. (A) Depletion of endogenous MOV10 has no effect on HIV-1 virus production. Stable MOV10 KD cells were produced by transducing HeLa or 293T cells with lentiviral vectors expressing either a non-silencing control shRNA or a MOV10-specific shRNA. HeLa or 293T non-silencing control and MOV10 KD cells were infected with VSV-G pseudotyped HIV-1NL4-3. Virus concentration in the medium was determined as described in Figure 1A. Cell lysates were analysed by immunoblotting with anti-p24Gag, anti-Hsp90 or anti-MOV10 antibodies, the latter of which was used to verify the MOV10 KD. (HeLa virus production p = 0.0611, 293T virus production p = 0.2007). (B) Silencing of endogenous MOV10 has no effect on HIV-1 virion infectivity. Virion infectivity was determined as described in Figure 1B. (HeLa infectivity p = 0.3080, 293T infectivity p = 0.4812). (C) Depleting endogenous MOV10 has no effect on spreading HIV-1 replication. Hut78 non-silencing control or MOV10 KD cells were infected with equal amounts of HIV-1NL4-3 and passaged every 2 days. Medium was harvested on days 2, 4, 6 and 8 and virus production was determined as described in Figure 1A. Cell lysates were analysed by immunoblotting with anti-MOV10 and anti-Hsp90 antibodies. (D) MOV10 silencing has no effect on the production of infectious SIVmac, MLV and M-PMV particles. 293T non-silencing control or MOV10 KD stable cells were transfected as described in Figure 1C for the production of HIV-1, SIVmac, MLV and M-PMV particles. Infectivity was determined as described in Figure 1C. (HIV-1 p = 0.1358, SIVmac p = 0.1040, MLV p = 0.4907, M- PMV p = 0.4919). For (A), (B) and (D) results are normalised to the non-silencing control, which is set at 100%. For (D) a single control bar set at 100% is graphed for simplicity. Values are the mean ± SD of 7 independent experiments for (A) and (B) or 3 independent experiments for (D). The data were analysed with an unpaired one- tailed t test.