SERINC5 co-expressed with HIV-1 Env or present in a target membrane destabilizes small fusion pores leading to their collapse.

SERINC5 is a multipass membrane protein that reduces infectivity of retroviruses and other enveloped viruses through incorporation into budding virions and inhibiting viral fusion. Several modes of anti-HIV activity of SERINC5 have been reported, including binding to Env glycoprotein, induction of conformational changes and destabilization of Env, as well as disruption of trans-membrane asymmetry of viral envelope. All these reported anti-HIV mechanisms involve SERINC5 incorporation into progeny virions, while there is very little information regarding a potential antiviral activity of SERINC5 in target cells. Here, we show that SERINC5 expressed in target cells efficiently inhibits fusion with cells expressing sensitive but not resistant HIV-1 strains. We show that this activity does not result from downregulation of CD4 or coreceptors on target cells or interference with the formation of ternary Env/CD4/coreceptor complexes. We further demonstrate that SERINC5 destabilizes small fusion pores causing their collapse and that this block can be rescued by incorporation of phosphatidylserine to either effector (Env-expressing) or target cells. Interestingly, we did not detect a significant reduction of HIV-1 pseudovirus fusion with SERINC5 expressing target cells, while virus-mediated cell-cell fusion (fusion-from-without) was inhibited, suggesting a potential role of virus' entry pathway in SERINC5 restriction. Collectively, our results reveal a novel mechanism of inhibition of HIV-1 fusion by SERINC5 through destabilization of small fusion pores in a manner that depends on lipid composition. Importance: SERINC5 incorporates into virions produced by infected cells and inhibits viral fusion with target cells through a poorly understood mechanism. Here, we show that SERINC5 blocks HIV-1 Env mediated cell fusion when expressed in either effector (Env-expressing) or target cells. Inhibition of Env-mediated fusion by SERINC5 expressed in target cells is not through reduction in receptor or coreceptor expression or interference with Env's ability to engage a requisite number of receptors and coreceptors. We demonstrate that the block of fusion is at a post-hemifusion stage of small fusion pores that collapse when SERINC5 is present in effector or target membrane and that this block is rescued by incorporation of specific anionic lipids, such as phosphatidylserine. These findings reveal a previously unappreciated mode of HIV-1 restriction through destabilization of small fusion pores that occurs irrespective of SERINC5 localization in either of fusing membranes.