Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV

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

Human IFITM proteins have previously been reported to restrict HIV cell-to-cell spread and replication [28, 29] . We next asked if there were any differences between primate IFITM restriction of cell-to-cell spread of HIV-1 (Fig 5) . This was important, because HIV-1 entry appeared not to be dramatically affected by the various IFITMs tested (Fig 4) . We utilized a method previously described in [54] that relies on an intron-regulated guassia luciferase (inGLuc) reporter gene that is expressed upon successful HIV infection of target cells [55] . Note that this assay measures only one-round of HIV-1 infection because it is based on an HIV-1 NL4.3 lentiviral vector. Donor Jurkat-inGLuc cells, either expressing or not expressing IFITMs, were infected with HIV-1 NL4.3 bearing VSV-G; 12 h post-infection, half of the cells were used to produce virus for cell-free infection while the other half were used for co-culture with target SupT1 cells expressing or not expressing IFITMs. Human IFITMs showed the greatest restriction (7-fold decrease for IFITM3) of HIV-1 cell-to-cell spread when human IFITM was expressed in both the target and donor cells (Fig 5A-5C ) [28, 29, 53] . Notably, while a similar trend was seen for nonhuman primate IFITMs (Fig 5A-5C) , there was a 21-fold decrease in HIV-1 cell-to-cell spread when both donor and target cells expressed OWM IFITM3 (3-fold greater compared to human IFITM3) (Fig 5A) . These results show that nonhuman primate IFITMs share similar, perhaps modestly enhanced, cell-to-cell restriction of HIV-1 infection as those of human IFITMs. We next examined the effects of primate IFITMs on HIV-1 replication by performing longterm replication assays using infectious WT NL4.3. Supernatants from cell culture were harvested every two days and quantified for viral production using reverse transcriptase (RT) assay. As shown in Fig 5D, the NL4.3 replication peaked on day 10 in both empty vector and human IFITM1-expressing SupT1 cells, whereas two nonhuman primate IFITM1 delayed the peak to day 14. Of note, human IFITM1 exhibited roughly half the viral production of empty vector control, consistent with the notion that its inhibitory effect on HIV-1 NL4.3 replication is modest [29] . We also found that the L'Hoest's OWM IFITM1 had a lower peak value compared to the gorilla counterpart (Fig 5D) . When IFITM3 protein was compared, we observed a peak shift of viral replication from day 10 to day 14 for both human and gorilla IFITM3 ( Fig 5E) . Strikingly, HIV-1 replication in L'Hoest's OWM IFITM3-expressing cells did not peak even after day 18 ( Fig 5E) . Overall, our results demonstrate that IFITM inhibition of HIV-1 replication is evolutionarily conserved, and that nonhuman primate IFITMs are more potent to inhibit HIV-1 replication than that those of human counterparts.