Coronaviruses have demonstrated the capacity for host-species switching, both in natural epidemics, such as SARS, and in the laboratory in cells from different species following passage, recombinant swapping of the spike protein, or following electroporation of genome RNA. Like other plus-strand RNA viruses, coronaviruses replicate in the host cell cytoplasm in association with modified cellular membranes. Coronaviruses cause profound modifications of cytoplasmic membranes, inducing a reticulovesicular network containing double- membrane vesicles as putative sites of viral RNA synthesis, also known as replication complexes. However, the viral and cellular determinants of replication complex formation and function are not known. In addition, infection with the coronavirus mouse hepatitis virus (MHV) is associated with continuous membrane ruffling and internalization, but the mechanisms and role of membrane ruffling in coronavirus replication is not known. The overall goals of the proposed research program is to elucidate the conserved and unique viral and cellular proteins, membranes, and pathways that mediate coronavirus induced cell membrane modifications and replication complex formation and establish the requirements for the membrane modifications in coronavirus replication. The three integrated aims of this proposal will use MHV and SARS-CoV in replication, cell imaging, proteomic and biochemical experiments to define the cellular and viral components of replication complexes, and establish the mechanisms and role of membrane ruffling in coronavirus replication. The results of these studies will identify new virus-host interactions that may be critical in the ability of coronaviruses to establish replication in multiple cell types and move between species. In addition the studies will likely define new viral targets for studies of virus host range and interference with virus replication. Finally the experiments may elucidate new pathways in host cell biology and interaction with intracellular pathogens.

Public Health Relevance

Coronaviruses are RNA viruses that replicate in the cytoplasm of the infected host cell and induce modifications to host cell membranes that serve as sites for viral RNA synthesis. The goal of the proposed research program is to define the process of coronavirus replicase protein targeting and viral RNA synthesis during coronavirus infection, identify cellular interacting proteins, and test the requirements for cellular proteins and pathways in successful coronavirus replication. The results of these studies will define common pathways of coronavirus replication, identify viral and cellular targets for studies of viral virulence, attenuation, and interference with coronavirus infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Virology - B Study Section (VIRB)
Program Officer
Park, Eun-Chung
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
Schools of Medicine
United States
Zip Code
Freeman, Megan Culler; Peek, Christopher T; Becker, Michelle M et al. (2014) Coronaviruses induce entry-independent, continuous macropinocytosis. MBio 5:e01340-14
Freeman, Megan Culler; Graham, Rachel L; Lu, Xiaotao et al. (2014) Coronavirus replicase-reporter fusions provide quantitative analysis of replication and replication complex formation. J Virol 88:5319-27
Beachboard, Dia C; Lu, Xiaotao; Baker, Susan C et al. (2013) Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive. Virology 435:210-3
Graham, Rachel L; Sparks, Jennifer S; Eckerle, Lance D et al. (2008) SARS coronavirus replicase proteins in pathogenesis. Virus Res 133:88-100
Sparks, Jennifer S; Donaldson, Eric F; Lu, Xiaotao et al. (2008) A novel mutation in murine hepatitis virus nsp5, the viral 3C-like proteinase, causes temperature-sensitive defects in viral growth and protein processing. J Virol 82:5999-6008
Zhao, Zijiang; Thackray, Larissa B; Miller, Brian C et al. (2007) Coronavirus replication does not require the autophagy gene ATG5. Autophagy 3:581-5
Sparks, Jennifer S; Lu, Xiaotao; Denison, Mark R (2007) Genetic analysis of Murine hepatitis virus nsp4 in virus replication. J Virol 81:12554-63
Denison, Mark R (2004) Severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update. Pediatr Infect Dis J 23:S207-14
Prentice, Erik; Jerome, W Gray; Yoshimori, Tamotsu et al. (2004) Coronavirus replication complex formation utilizes components of cellular autophagy. J Biol Chem 279:10136-41
Brockway, Sarah M; Lu, Xiao Tao; Peters, Timothy R et al. (2004) Intracellular localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus replication. J Virol 78:11551-62

Showing the most recent 10 out of 11 publications