SAM mutants increased HIV-1 production in HEK293T. (A) Schematic structure of the viperin protein showing N- and C-terminal truncations and SAM domain mutants. (B-D) HEK293T were cotransfected with pWT/BaL proviral HIV-1 DNA and a panel of either WT viperin or SAM mutants (S1, S2, S3, S1 + S2 + S3; individually) or 5′ or 3′ truncation mutants (5′17, 5′33, 5′50, 5′100, 3′17, 3′33, 3′50, 3′100; individually) or plasmid control (pLNCX2 vector). (B) SAM domain mutations significantly reverse viperin inhibition of HIV-1. Viral production was quantified on TZM-bl as in Figure 2C. Histograms show relative fold changes in the percentage of HIV-1 infection, which is indicative of HIV production from the HEK293T cells. The mean data from 3 experiments are shown with SE bars. (C) WT viperin and SAM mutants do not diminish the proportion of HIV-1–infected cells. After 48 to 72 hours after transfection, staining for intracellular p24 was performed. The percentage of intracellular p24 cells was determined by gating against non-pBaL–transfected cultures by flow cytometry. The mean data from 4 experiments are shown with SE bars. (D) SAM domain mutants up-regulate total extracellular p24 released in the culture supernatants compared with WT viperin. Supernatants from HEK293T cell cotransfectants were collected at 72 hours after transfection, and levels of extracellular virus were quantified by p24 ELISA (XpressBio) according to the manufacturer's instructions. The mean data from 4 experiments are shown with SE bars.