Lai RP, Yan J, Heeney J, McClure MO, Gottlinger H, Luban J, Pizzato M

Lai RP, Yan J, Heeney J, McClure MO, Gottlinger H, Luban J, Pizzato M. the maintenance of high viral loads in patients. However, the underlying molecular mechanism remained elusive until the recent discovery of the antiviral activity of SERINC5. SERINC5 profoundly inhibits HIV-1 but is antagonized by Nef, which prevents the incorporation of SERINC5 into viral particles. Here, we show that HIV-1 Env, but not Nef, is able to resist high levels of SERINC5 without excluding SERINC5 from incorporation into viral particles. However, the virion-associated SERINC5 renders HIV-1 more sensitive to some broadly neutralizing antibodies. It is possible that, under the pressure of some neutralizing antibodies values were calculated using the Wilcoxon signed-rank test. (Right) The levels of Nef proteins were assessed by Western blotting. (F) The Nef proteins of strains NL4-3, AD8-1, and YU-2 did not overcome the inhibition by ectopic SERINC5. NL4-3(Nef) DNA was cotransfected with SERINC5 DNA as well as plasmid DNA that expressed NefNL4-3, NefAD8-1 or NefYU-2. (Left) The infectivity of the viruses was determined by infecting TZM-bl cells. (Right) The fold inhibition by SERINC5 from three independent transfections is summarized. *, < 0.05; **, < 0.01; NS, not significant. It is possible that the observed resistance phenotype of AD8-1 and YU-2 is a result of the ability of their Nef proteins to counter high levels of SERINC5. To test this, we cloned the Nef genes of strains NL4-3, AD8-1, and YU-2 into expression vectors and first tested their function in downregulating cell surface CD4 (Fig. 1D and ?andE).E). All three Nef proteins were expressed to similar levels (Fig. 1E). The Nef proteins of both strain Cenicriviroc Mesylate NL4-3 (NefNL4-3) and strain AD8-1 (NefAD8-1) were equally effective in diminishing cell surface CD4, whereas the Nef of strain YU-2 (NefYU-2) was less effective, likely as a result of a sequence inserted in its N-terminal region (Fig. 1D and ?andE)E) (19). We then cotransfected these Nef DNA clones with NL4-3(Nef) and SERINC5 DNA and observed Cenicriviroc Mesylate that none of the three Nef proteins were able to markedly overcome the inhibition by the overexpressed SERINC5 (Fig. 1F). These data suggest that AD8-1 and YU-2 have a Nef-independent mechanism to resist high levels of ectopic SERICN5. The V3 loop determines the ability of HIV-1 Env to counter SERINC5. The Env protein of some HIV-1 strains has been reported to resist SERINC5 inhibition (16, 17). We therefore measured SERINC5 inhibition of an NL(AD8Env) virus that had the NL4-3 Env replaced by the Env of AD8-1 (20). The results showed that NL(AD8Env) was as resistant to SERINC5 as AD8-1, suggesting that the AD8-1 Env is sufficient to render the otherwise sensitive NL4-3 virus resistant to high levels of SERINC5 (Fig. 2A). The V1 and V2 loops of Env have been shown to contribute to SERINC5 resistance (17). Cenicriviroc Mesylate Accordingly, we replaced the V1, V2, and V3 loop sequences in NL4-3 Env with their counterparts in the AD8-1 Env. The resultant viruses, NL(AD8V1V2V3), which had all three V loops replaced, and NL(AD8V3), which had only the V3 loop replaced, were resistant to the overexpressed SERINC5 (Fig. 2A). The NL(AD8V1) and NL(AD8V2) viruses were not viable and were not tested for SERINC5 inhibition. We further investigated the role of the Cenicriviroc Mesylate V3 loop in resisting SERINC5 inhibition by inserting into NL4-3 Env the V3 loop sequences from HIV-1 strains YU-2, RHPA, WITO, and THRO. The Mouse monoclonal to CRTC1 phenotypes of these V3 loop chimeric viruses, NL(YU2V3), NL(RHPAV3), NL(WITOV3), and NL(THROV3), respectively, recapitulated the SERINC5 resistance phenotype of the parental viruses from which the V3 loop was derived (Fig. 2B). We also generated V3 chimeric viruses that had a nonfunctional mutant NefG2A protein and observed that these chimeric viruses were also resistant to SERINC5 inhibition (Fig. 2C). Open in a separate window FIG 2 HIV-1 Env resists SERINC5 inhibition. (A) SERINC5 DNA was cotransfected with NL4-3 DNA that bears either the Env of AD8-1 (AD8Env), the V1, V2, and V3 loop sequences of AD8-1 Env (AD8V1V2V3), or only the V3 loop sequence of AD8-1 Env (AD8V3). (Left) The infectivity of these.