Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV

Wilkins, Jordan; Zheng, Yi-Min; Yu, Jingyou; Liang, Chen; Liu, Shan-Lu

To ensure that endogenous IFITM3 of nonhuman primate cell origin contributed to the restriction of HIV-1, we knocked down IFITM3 in gorilla fibroblasts and examined the effect on HIV-1 short-term replication (Fig 6) . Gorilla IFITM3 was knocked down using a lentiviral small hairpin RNA against IFITM3. Expression of gorilla IFITM3 was reduced compared to that of a non-specific control shRNA (Fig 6A) . Gorilla cells were infected with HIV-1 NL4.3 psuedotyped with VSV-G to ensure entry. After a single round of replication, supernatants containing newly generated virus were harvested and examined for RT activity and by infecting HeLa-TZM-bl cells (Fig 6B and 6C, respectively) . There was more than a 2-fold increase in RT activity of viral supernatants from IFITM3 knockdown cells (Fig 6B) . Consistently, the infectivity of produced virus was also increased when gorilla IFITM3 was knocked down (Fig 6C) . These results support the conclusion that the ability of endogenous nonhuman primate IFITMs to restrict lentiviruses is ancestrally conserved. Analysis of protein-coding nucleotide substitutions can provide insight into the type of selective pressure on proteins over time [56] . Nucleotide substitutions that result in a change in the amino acid sequence are referred to as nonsynonymous (dN) while silent changes are considered synonymous (dS). A ratio of nonsynonymous over synonymous substitutions (dN/dS) can be used to detect for positive, neutral, or purifying selection (also referred to as negative selection) [56] . To infer what type of selective pressure the IFITM proteins were under, we analyzed 9 primate IFITM1 or 9 primate IFITM3 sequences, including those of AGM we recently described [49] using Datamonkey [57] [58] [59] (Table 2 ) and MEGA6 software [44] (S1 Fig). In both cases, analysis of nucleotide sequences from IFITM1 and IFITM3 showed that the values of dN were much less than dS. Expanding the primate IFITM sequences to include additional IFITMs of other species from GenBank yielded similar results (data not shown), suggesting that IFITM proteins are under purifying selection (Table 2 and S1 Fig) . Taken together, these results suggest that the primary amino acid sequence of the IFITM proteins are crucial for their biological functions and that most nonsynonymous changes could be deleterious to IFITMs.