Coronavirus Replicase-Reporter Fusions Provide Quantitative Analysis of Replication and Replication Complex Formation

Public Deposited
Analytics

Downloadable Content

Download PDF
Request Version for Screen Reader
Creator
  • Megan Culler Freeman
    • Other Affiliation: Departments of Pathology, Microbiology, and Immunology, and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA
  • Rachel L. Graham
    • Other Affiliation: Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA
  • Xiaotao Lu
    • Other Affiliation: Departments of Pediatrics and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA
  • Christopher T. Peek
    • Other Affiliation: Departments of Pediatrics and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA
  • Mark R. Denison
    • Other Affiliation: Departments of Pediatrics and Pathology, Microbiology, and Immunology, and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA
Abstract
  • The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication.
Date of publication
Keyword
DOI
Related resource URL
Resource type
  • Article
Rights statement
  • In Copyright
Journal title
  • Journal of Virology
Journal volume
  • 88
Journal issue
  • 10
Page start
  • 5319
Page end
  • 5327
Language
  • English
ISSN
  • 0022-538X
  • 1098-5514
  • 1070-6321
Publisher
  • American Society for Microbiology

Relations

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details zjv5319.pdf 2020-04-06 Public Download