CD4 Expression and Env Conformation Are Critical for HIV-1 Restriction by SERINC5

Abstract

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, tier 1 HIV-1 Env proteins are sensitive to SERINC5, whereas the majority of tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing tier 1 NL Env with tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of the two viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interactions. A much greater level of NL Env-SERINC5 interactions was detected than was AD8 Env-SERINC5 interactions, which was further validated by immunoprecipitation assays. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex when CD4 was not expressed. However, when CD4 was expressed, SERINC5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because tier 1 and tier 2/3 Env trimers have open and closed conformations, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication. IMPORTANCE Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env associations and Env-SERINC5 interactions in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

“…SERINC5 strongly affected ΔNef and, to a lower extent, WT infectivity (39). CD4 expression has been recently suggested as modulating the effect of SERINC5 on Env fusion (43). We produced fluorescent NL4-3 and THRO virions in 293T or 293T cells stably expressing CD4 (293T-CD4), with or without transient transfection of SERINC5.…”

“…The exact mechanism used by SERINC5 to restrict HIV-1 entry is not completely understood. A correlation between Env sensitivity to antibody neutralization and to SERINC5 has been reported, suggesting that SERINC5 activity may depend on Env conformations (41)(42)(43).…”

“…SERINC5-mediated viral restriction also depends on Env, and tier 1 Env appears to be more sensitive than tier 2/3 Env (41,42). It has been suggested that SERINC5 selectively targets open Env trimers (43).…”

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

“…This hypothesis is consistent with the genetic studies that mapped the determinants of SERINC5 sensitivity to the variable loops in gp120 20,29 , as amino acid changes in the variable loops can modulate the accessibility of MPER 30 . Moreover, the interaction with CD4, which triggers an open HIV-1 Env conformation and increases MPER accessibility 31 , rendered tier 2 and 3 viral isolates sensitive to restriction by SERINC5 21 . At present, we cannot exclude other possibilities, such as interactions with viral glycans and/or conformational movements of the spike that would enable direct SERINC5 contacts with gp120.…”

“…HIV-1 Env is highly variable, and individual viral isolates display vastly different sensitivities to neutralizing antibodies and restriction by SERINC5. Thus, while SERINC5 can ablate infectivity of the HIV-1 clone NL4-3, it has only a modest effect on the tier 2 and 3 HIV-1 isolates, such as JRFL 2,3,10,20,21 . Nevertheless, JRFL becomes considerably more sensitive to some neutralizing antibodies and the CCR5 antagonist Maraviroc in the presence of SERINC5, suggesting that the restriction factor exerts a direct effect on the conformation of HIV-1 Env 10,20 .…”