SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue
Fig 8
SERINC5 antagonism does not enhance viral replication in primary human CD4+ T cell cultures.
(A) Stimulated primary CD4+ T cells were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were maintained in culture for up to 10 days post-transduction under static conditions (left panel) or with agitation (right panel) to limit cell-to-cell viral spread. Every 2 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield by TZM-bl reporter cell assay and CD4+ T cell infection rates were assessed by intracellular p24 staining followed by flow cytometric analysis. Data shown represents measurements obtained from 3 donors (mean ±SEM). (B) Correlation between the infection rates of TZM-bl and primary CD4+ T cells infected with HIV-1 IRES-eGFP nef recombinants produced in the presence of transiently expressed SERINC5 in HEK293T cells. (C) The infectious virus and p24 antigen yields at 4, 6 and 8 days post-infection in the standard CD4+ T cell infection experiments shown in panel A were determined by TZM-bl infection and p24 antigen ELISA, respectively. Virion infectivity normalized for p24 content is shown relative to the nef-defective HIV-1 construct (100%). Shown are average values obtained at three time points for the three blood donors (+SEM). *p < 0.05; **p < 0.01.