SERINC5 Restricts HIV-1 Infectivity by Promoting Conformational Changes and Accelerating Functional Inactivation of Env.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.