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. 2014 Dec 16;89(1):535–544. doi: 10.1128/JVI.02751-14

FIG 7.

FIG 7

oBST2B reduces infectivity of MLV-based vectors pseudotyped by JSRV Env or VSV-G glycoproteins. (A) Representative Western blots of cellular extracts (lysates) of CPT-Tert transfected with 1 μg of the expression plasmid for oBST2B-HA [CPT (1 μg)] or 293T cells stably expressing oBST2B-HA (293T/B-HA). oBST2B-HA was detected using an anti-HA antibody. α-Tubulin (α-Tub) was used as a sample-loading control. (B) 293T/B-HA cells were analyzed by confocal microscopy using antibodies to the HA epitope and cadherins, as indicated in the panel. The scale bar represents 10 μm. (C) Western blots of concentrated viral particles recovered from the supernatants of 293T or 293T/B-HA cells (indicated by a minus or a plus sign, respectively) transfected with the expression plasmids in the amounts (5 μg, 2 μg, or 0.8 μg) reported above the blots. Blots were incubated with anti-MLV-CA and anti-JSRV-SU or anti-VSV-G antibodies, as indicated. Each experiment was repeated independently three times, and representative Western blots are shown. Viral-particle release and Env incorporation of JSRVpp-2 μg and JSRVpp-0.8 μg produced in 293T and 293T/B-HA cells were assessed by chemifluorescence. Values for CA and SU expression in 293T/B-HA cells were related to values obtained in 293T control cells (arbitrarily set as 100%). The graph represents the SU/CA ratios of JSRVpp-2 μg and JSRVpp-0.8 μg produced in 293T/B-HA cells. Note that there is a statistically significant difference (P = 0.01, calculated by one-way ANOVA) in 293T/B-HA cells that depends on the amount of JSRV Env expression plasmid transfected. Open circles indicate ratio values, while black horizontal bars represent the mean ratio values of the data obtained. (D) NoEnvpp-, JSRVpp-, and VSVpp-infected CPT-Tert cells were analyzed by fluorescence-activated cell sorter (FACS) at 72 h postinfection to quantify the percentage of GFP-positive cells. The percentage of cells infected with JSRVpp and VSVpp produced in control 293T cells was designated 100% of infectivity. Experiments were performed independently at least three times with two biological replicates for each CPT-Tert infection. Bars indicate standard errors.