Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques
Fig 5
Sequence changes in Nef restore resistance to tetherin and SERINC5.
(A) The virus population in plasma of animals infected with SIVmac239AAA was sequenced at the indicated time points post-infection. The predicted amino acid sequences in the flexible loop region of Nef are aligned to the corresponding Nef sequences of SIVmac239AAA and SIVmac239. Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code. (B & C) The Nef variants observed at 22–24 weeks PI in SIVmac239AAA-infected animals were tested for the ability to counteract tetherin (B) and SERINC5 (C). (B) 293T cells were co-transfected with SIVmac239Δnef together with constructs expressing the indicated Nef variants and rhesus macaque tetherin. Percent maximal virus release was calculated from the accumulation of SIV p27 in the culture supernatant of cells transfected with tetherin relative to cells transfected with empty vector. Differences in virus release were corroborated by western blot analysis as described in Fig 1. (C) C8166-SEAP cells were infected with Nef trans-complemented virus produced by co-transfection of parental JTAg cells (SERINC3+5+) with SIVmac239Δnef and the indicated Nef variants. Infectivity was measured as described in Fig 3 and Nef expression was confirmed by western blot analysis. (B & C) Error bars indicate standard deviation of the mean for at least four independent experiments and significant differences relative to NefAAA are indicated by asterisks (*p<0.05, ** p<0.01, *** & p<0.001, two-tailed unpaired t-test with Welch’s correction in case of unequal variance).