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Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus
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Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

Mark A Clementz1, Amornrat Kanjanahaluethai, Timothy E O'Brien

  • 1Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, IL 60153 USA.

Virology
|February 26, 2008
PubMed

Insights

Murine coronavirus nonstructural protein 4 (nsp4) is essential for double membrane vesicle (DMV) assembly. Mutations in nsp4 disrupt DMV formation and viral replication, highlighting its critical role in coronavirus replication.

Area of Science:

  • Virology
  • Molecular Biology
  • Cell Biology

Background:

  • Coronaviruses are positive-strand RNA viruses replicating in the cytoplasm.
  • Viral replication involves a membrane-associated replicase complex assembled on double membrane vesicles (DMVs).
  • Nonstructural protein 4 (nsp4) is a putative replicase anchor in coronavirus replication.

Purpose of the Study:

  • To investigate the role of nsp4 in the assembly of murine coronavirus DMVs.
  • To analyze the impact of specific nsp4 mutations on viral replication and DMV formation.

Main Methods:

  • Reverse genetics was used to generate infectious clone viruses (icv) with nsp4 mutations.
  • Mutations included alanine substitutions at glycosylation sites (N176A, N237A) and a temperature-sensitive mutation (N258T).
  • Viral replication and protein localization were analyzed in infected cells at permissive and non-permissive temperatures.

Main Results:

  • The nsp4-N237A mutation was lethal.
  • The nsp4-N258T mutation resulted in a temperature-sensitive virus (Alb ts6 icv) with reduced viral replication at non-permissive temperatures.
  • Alb ts6 icv-infected cells showed a dramatic reduction in DMVs, with partial relocalization of nsp4 and nsp3 to mitochondria at the non-permissive temperature.

Conclusions:

  • nsp4 plays a critical role in directing the assembly of coronavirus DMVs.
  • Disruption of nsp4 function impairs DMV formation and viral replication.
  • nsp4's function is essential for the proper assembly of the viral replicase complex and subsequent viral propagation.

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