Leucine-rich repeat-containing G protein–coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein

Na Zhang,Yinglei Wei,Lili Hou,Min Qian,Stefan Siwko,Mingyao Liu,Qingqing Xiong,Honghui Han,Nannan Wu,Andrea Cimarelli,Bing Du,Binghe Tan,Hongjun Huang,Yan Yan,Jianrong Xu

doi:10.1074/jbc.m117.802090

Abstract

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.

Highlights

The distribution of vesicular stomatitis virus (VSV) in leucine-rich repeat-containing G protein– coupled receptor 4 (Lgr4)-deficient olfactory bulbs was reduced significantly compared with wild-type mice through intranasal infection. It suggested that Lgr4 plays a nonredundant role in the VSV infection mouse model
VSV-induced cell death (Fig. 1B) was significantly reduced in Lgr4-deficient mouse embryonic fibroblast (MEF) cells compared with wild-type cells
The condensed nucleocapsid of VSV is surrounded by a lipid bilayer containing the viral glycoprotein G that constitutes the spikes that protrude from the viral surface

Summary

Results

To investigate the significance of Lgr in viral infection, we intranasally infected Lgr4ϩ/ϩ and Lgr4Ϫ/Ϫ mice with VSV (1 ϫ 106 pfu) for 24 h. The distribution of VSV in Lgr4-deficient olfactory bulbs was reduced significantly compared with wild-type mice through intranasal infection It suggested that Lgr plays a nonredundant role in the VSV infection mouse model. Overexpression of Lgr in RAW 264.7 cells significantly increased the GFP fluorescence intensity (Fig. 3E) These data confirmed that Lgr increased VSV-G-mediated infection. Because Lgr deficiency significantly reduced VSV infection even in MEFs where endocytosis was blocked by Pitstop treatment, it is likely that Lgr functions at a different stage of VSV infection than endocytosis. Taken together, these results implied that Lgr has a major role in very early stages of VSV infection, such as viral attachment. Ing endogenous Lgr could be a potential way to prevent VSV infection

Discussion

Experimental procedures

Ethics statement