Coronaviruses (CoV) are associated with severe diseases as demonstrated by the 2003 severe acute respiratory syndrome (SARS)-CoV epidemic. One of the critical steps of infection involves viral mRNA mediated recoding of gene expression;a -1 frameshifting event that occurs during translation. It is this elegant mechanism that allows the ribosome to bypass a stop codon and synthesize viral enzymatic proteins. Furthermore, the frequency by which this event occurs is important for efficient viral infectivity, and is regulated by domains in the translating mRNA (in the case of the SARS-CoV, this domain is a pseudoknot). Although aspects of frameshifting have received considerable attention, an understanding of the RNA structures and structural rearrangements that influence the efficiency is lacking. Our preliminary studies indicate that retroviral gene expression is regulated by a dynamic, proton-driven equilibrium between an active, read-through permissive, and an inactive pseudoknot conformation. This proposal aims to gain a complete structural and mechanistic understanding of the frameshifting frequency in SARS-CoV by combining structural studies with biochemical and in vivo experiments.
Our aims will be: (#1) to understand the basis for how the recoding frequency is maintained, (#2) to determine the structures of the mRNA signal in both configurations: permissive and non-permissive for frameshifting and (#3) to engineer structure-guided mutants to test our equilibrium model.

Public Health Relevance

The Severe Acute Respiratory Syndrome (SARS) Coronavirus is known to cause fatal respiratory disease. Our study will help determine the mechanism involved in programmed ribosomal frameshifting, which is required for efficient viral infectivity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI104711-02
Application #
8616027
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Stemmy, Erik J
Project Start
2013-02-07
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
2
Fiscal Year
2014
Total Cost
$342,225
Indirect Cost
$139,725
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138