Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

Rebecca S. Brown,Dimitrios G. Anastasakis,Markus Hafner,Margaret Kielian

doi:10.1038/s41467-020-18447-z

Abstract

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

Highlights

The alphavirus capsid protein (Cp) selectively packages genomic RNA into the viral nucleocapsid to produce infectious virus
We observed no difference in virus or NC morphology by transmission electron microscopy (Supplementary Fig. 1b), or in the expression or localization of biotinylated Cp-mAVI vs. CpWT in infected cells (Fig. 1b and Supplementary Fig. 1c–e.) No change was detected in Semliki Forest virus (SFV)-mAVI vs. WT specific infectivity as calculated by comparing the number of infectious particles to total particles (PFU:E2 ratio; Fig. 1c), indicating accurate packaging of the genomic RNA (gRNA) by Cp-mAVI
Using the biotin handle on Cp, we optimized retrieval conditions with Streptavidin beads to efficiently and pull-down Cp. Under these stringent conditions, Cp was retrieved from infected BirA cells only when biotin was present in the culture medium (Fig. 1d)

Summary

Introduction

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. 1234567890():,; Alphaviruses are enveloped, highly organized RNA viruses that include a number of important human pathogens such as Chikungunya virus (CHIKV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV)[1]. These viruses are transmitted by mosquito vectors and can infect a wide variety of mammalian and avian species. An internal promoter drives the production of a smaller

Methods

Results

Conclusion