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. Author manuscript; available in PMC: 2014 Jul 21.

Published in final edited form as: Virology. 2011 Aug 25;419(1):24–42. doi: 10.1016/j.virol.2011.07.017

Fig. 6 — Inhibition of SHIVΔVif and HIVΔVif virion infectivity by rhA3A. Panel A. RhA3A restricts SHIVΔvif. 293 cells were co-transfected with 1 μg wild-type SHIV or SHIVΔvif molecular clones and 0.5 μg plasmids expressing either empty vector (pcDNA3.1), rhA3A, hA3A, or rhA3G. At 48 h, the culture medium was harvested and virion infectivity measured by taking the ratio of infectious viral titers on TZM-bl cells. Shown is the percentage virion infectivity with the empty plasmid control normalized to 100%. Panel B. 293 cells were transfected with wild-type SHIV or SHIVΔvif molecular clones and HA-tagged rhA3A, hA3A, or rhA3G. At 48 h, the culture medium was collected, clarified by low speed centrifugation, and concentrated by ultracentrifugation through a 20%/60% (w/v) step-gradient. Each sample was resuspended in 2X sample reducing buffer, boiled, and the proteins were separated by SDS-PAGE. The presence of rhA3G, hA3A, or rhA3A was detected by Western blot using an antibody directed against the HA-tag (upper) and the presence of p27 using an antibody directed against p27 (middle). The lower panel are 293 cells transfected with vectors expressing rhA3G, hA3A, rhA3A or pcDNA3.1(+) vector in the absence of the viral genome. Panel C. RhA3A restricts HIV-1Δvif virions. HIV-1 or HIV-1Δvif genomes were co-transfected with HA-tagged hA3A, hA3G or rhA3A as described for the SHIV experiments in Panel A. Shown is the percentage virion infectivity with the empty plasmid control normalized to 100%. Panel D. Undetectable rhA3A in HIV-1Δvif virions. Virions were purified as described for the SHIV experiments in Panel B, and hA3G, hA3A and rhA3A incorporation were detected by Western blot using an anti-HA antibody, normalizing for Gag p24 levels. The lower panel are 293 cells transfected with vectors expressing rhA3G, hA3A, rhA3A or pcDNA3.1(+) vector in the absence of the viral genome. Mean values in triplicate experiments are shown, and statistical differences with the wild-type control were evaluated using a two-tailed Student’s t-test, with p<0.05 (▲) considered significant.

Fig. 6 — Inhibition of SHIVΔVif and HIVΔVif virion infectivity by rhA3A. Panel A. RhA3A restricts SHIVΔvif. 293 cells were co-transfected with 1 μg wild-type SHIV or SHIVΔvif molecular clones and 0.5 μg plasmids expressing either empty vector (pcDNA3.1), rhA3A, hA3A, or rhA3G. At 48 h, the culture medium was harvested and virion infectivity measured by taking the ratio of infectious viral titers on TZM-bl cells. Shown is the percentage virion infectivity with the empty plasmid control normalized to 100%. Panel B. 293 cells were transfected with wild-type SHIV or SHIVΔvif molecular clones and HA-tagged rhA3A, hA3A, or rhA3G. At 48 h, the culture medium was collected, clarified by low speed centrifugation, and concentrated by ultracentrifugation through a 20%/60% (w/v) step-gradient. Each sample was resuspended in 2X sample reducing buffer, boiled, and the proteins were separated by SDS-PAGE. The presence of rhA3G, hA3A, or rhA3A was detected by Western blot using an antibody directed against the HA-tag (upper) and the presence of p27 using an antibody directed against p27 (middle). The lower panel are 293 cells transfected with vectors expressing rhA3G, hA3A, rhA3A or pcDNA3.1(+) vector in the absence of the viral genome. Panel C. RhA3A restricts HIV-1Δvif virions. HIV-1 or HIV-1Δvif genomes were co-transfected with HA-tagged hA3A, hA3G or rhA3A as described for the SHIV experiments in Panel A. Shown is the percentage virion infectivity with the empty plasmid control normalized to 100%. Panel D. Undetectable rhA3A in HIV-1Δvif virions. Virions were purified as described for the SHIV experiments in Panel B, and hA3G, hA3A and rhA3A incorporation were detected by Western blot using an anti-HA antibody, normalizing for Gag p24 levels. The lower panel are 293 cells transfected with vectors expressing rhA3G, hA3A, rhA3A or pcDNA3.1(+) vector in the absence of the viral genome. Mean values in triplicate experiments are shown, and statistical differences with the wild-type control were evaluated using a two-tailed Student’s t-test, with p<0.05 (▲) considered significant.