Serine incorporator protein 5 (SERINC5) is the host antiretroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. We also show that SERINC5 and SERINC2 also form clusters on single virions. Unexpectedly, Env and SERINC molecules exhibited poor codistribution on virions, as evidenced by much greater Env-SERINC pairwise distances compared to Env-Env distances. This observation is inconsistent with the previously reported interaction between Env and SERINC5 and suggests an indirect effect of SERINC5 on Env cluster formation. Collectively, our results reveal a multifaceted mechanism of SERINC5-mediated restriction of HIV-1 fusion that, aside from the effects on individual Env trimers, involves disruption of Env clusters, which likely serve as sites of viral fusion with target cells.
