TY - JOUR
T1 - Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA
AU - Brown, Rebecca S.
AU - Anastasakis, Dimitrios G.
AU - Hafner, Markus
AU - Kielian, Margaret
N1 - Funding Information:
We thank all of the members of the Kielian lab and Dimitrios Zattas for helpful discussions and comments on the manuscript. We thank Matthew Angeliadis for technical assistance. We thank the Einstein Analytical Imaging Facility and facility members Xheni Nishku and Timothy Mendez for training and technical assistance on the electron microscope. We thank Susan Buhl and Matthew Scharff for technical assistance and use of their plate reader. This work was supported by grants to M.K. from the National Institute of General Medical Sciences (R01-GM057454) and the National Institute of Allergy and Infectious Diseases (R01-AI075647) and by Cancer Center Core Support Grant NIH/NCI P30-CA13330. R.S.B. was supported by an NIH NRSA (F32-GM122450) postdoctoral fellowship and the Charles H. Revson Senior Fellowship in Biomedical Sciences. M.H. and D.G.A are supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences, the National Institute of Allergy and Infectious Diseases, National Institute of Arthritis and Musculoskeletal and Skin Diseases, or the National Institutes of Health. This study was made possible (in part) by funds granted by the Charles H. Revson Foundation. The statements made and views expressed, however, are solely the responsibility of the authors.
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-020-18447-z
DO - 10.1038/s41467-020-18447-z
M3 - Article
C2 - 32943634
AN - SCOPUS:85091192920
SN - 2041-1723
VL - 11
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 4693
ER -