3D Superresolution Studies of the Effect of SERINC5 on Env Glycoprotein Distribution on HIV-1 Particles

Abstract

HIV-1 envelope glycoprotein (Env) initiates viral fusion to target cells following the sequential interactions with CD4 and coreceptors. It has been shown that, following HIV-1 maturation, Env is unevenly distributed on the virus surface and suggested that such Env clustering facilitates viral fusion by increasing the local density of these proteins, which may act cooperatively to form a fusion pore. Here we performed super-resolution imaging of single viruses to determine if clustering of Env is disrupted by the recently discovered HIV-1 restriction factor, SERINC5, which incorporates into virions and inhibits viral fusion. A SERINC5-sensitive HIV-1 strain HXB2 was used in these experiments. The distribution of HXB2 Env and SERINC5 on single viruses was examined by 2D (dSTORM or STED) and 3D (iPALM) super-resolution microscopy. Protein clustering in single virions was analyzed by pixel-based intensity thresholding on STED images or by point coordinate-based DBSCAN algorithm on single-molecule localizations acquired by dSTORM or iPALM. Control viruses contained SERINC2 that lacks antiviral activity. Importantly, a smaller fraction of Env glycoproteins was found in clusters on SERINC5-containing viruses compared to control virions, while SERINC2-containing viruses tend to form more clusters that control viruses. With 3D/2D super-resolution fluorescence imaging on single viruses, our findings support the model according to which SERINC5 exerts it antiviral activity, at least in part, by disfavoring Env clustering and thus reducing the probability of fusion/infection. This work was supported by the NIGMS R01 grant GM054787 to GBM.