Coronaviruses are a family of enveloped RNA viruses that cause respiratory, enteric, and neurologic diseases in mammalian and avian hosts. In humans, four coronaviruses are responsible for common upper respiratory tract infections;a fifth human coronavirus is the causative agent of severe acute respiratory syndrome (SARS), which has the potential to re-emerge into the population from animal reservoirs. The overall goal of this proposal is to delineate mechanisms that are critical to multiple phases of coronavirus replication, using the prototype coronavirus mouse hepatitis virus (MHV). Coronaviruses have the largest genomes of all RNA viruses and their basic molecular biology is consequently intricate. There thus remain numerous gaps in our knowledge of the essential events at the earliest stages of infection, following entry of the viral nucleocapsid into the host cell, and at late stages of infection, when progeny envelope proteins and nucleocapids combine for budding. To manipulate the genomes of coronaviruses, we developed the earliest reverse genetic system, via targeted RNA recombination. This robust and versatile technique has been an indispensable tool for analyses of MHV structural proteins. More recently, full-length coronavirus cDNA systems have also become available, allowing access to the viral replicase. These powerful reverse genetic methodologies, together with complementary biochemical and molecular biological approaches, will be employed to accomplish three specific aims. (1) We will investigate a newly discovered critical interaction between the nucleocapsid (N) protein and a component of the viral replication-transcription complex (nsp3). This will entail identification of the particular modules of the huge nsp3 molecule that are essential for viral replication. (2) We will elucidate the crucial role of N protein in the initiation of coronavirus infection, which, we hypothesize, is to deliver the viral genome to the nascent replication-transcription complex. Moreover, we will learn how this role is modulated by phosphorylation of N protein. (3) We will further dissect the network of structural protein interactions among N protein, the membrane (M) protein, and the small envelope (E) protein that are essential to virion assembly. These efforts will build upon our discovery of unusual M protein variants that evolve in E deletion mutants, and will use interspecies M protein chimeras to probe M-M and M-N interactions. Additionally, we will explore the role of the genomic packaging signal in the selective incorporation of the nucleocapsid into virions.

Public Health Relevance

An understanding of the molecular biology of coronaviruses is critical for their control and prophylaxis. This project will provide fundamental insights into essential elements of the coronavirus infectious cycle. The proposed studies will identify prospective targets for antiviral chemotherapy and enable potential strategies to manipulate these pathogenic agents for vaccine design.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI064603-09
Application #
8631033
Study Section
Virology - A Study Section (VIRA)
Program Officer
Stemmy, Erik J
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
City
Menands
State
NY
Country
United States
Zip Code
12204
Kuo, Lili; Koetzner, Cheri A; Hurst, Kelley R et al. (2014) Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein. J Virol 88:4451-65
Yao, Qianqian; Masters, Paul S; Ye, Rong (2013) Negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus. Virology 442:74-81
Hurst, Kelley R; Koetzner, Cheri A; Masters, Paul S (2013) Characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein 3 of the viral replicase-transcriptase complex. J Virol 87:9159-72
Kuo, Lili; Masters, Paul S (2013) Functional analysis of the murine coronavirus genomic RNA packaging signal. J Virol 87:5182-92
Kuo, Lili; Masters, Paul S (2010) Evolved variants of the membrane protein can partially replace the envelope protein in murine coronavirus assembly. J Virol 84:12872-85
Hurst, Kelley R; Koetzner, Cheri A; Masters, Paul S (2009) Identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein. J Virol 83:7221-34
Kuo, Lili; Hurst, Kelley R; Masters, Paul S (2007) Exceptional flexibility in the sequence requirements for coronavirus small envelope protein function. J Virol 81:2249-62
Masters, Paul S (2006) The molecular biology of coronaviruses. Adv Virus Res 66:193-292
Hurst, Kelley R; Kuo, Lili; Koetzner, Cheri A et al. (2005) A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein. J Virol 79:13285-97