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Related Experiment Videos

Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA

Y F Wang1, S G Sawicki, D L Sawicki

  • 1Department of Microbiology, Medical College of Ohio, Toledo 43699.

Journal of Virology
|October 1, 1994
PubMed
Summary

Mutations in Sindbis virus nsP3 protein disrupt early negative-strand RNA synthesis by preventing the formation of the initial replication complex. This finding reveals a crucial role for nsP3 in alphavirus replication.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Alphavirus replication relies on nonstructural proteins (nsPs) for RNA synthesis.
  • Mutants like Sindbis virus HR ts4 and ts7 exhibit defects in negative-strand RNA synthesis.
  • Previous studies mapped mutations in complementation group A to nsP2.

Purpose of the Study:

  • To identify the specific mutation in Sindbis virus HR ts4 and its effect on viral replication.
  • To elucidate the role of nsP3 in the early stages of alphavirus negative-strand RNA synthesis.
  • To understand the functional relationship between nsP2 and nsP3 in the alphavirus replication complex.

Main Methods:

  • Genetic mapping of mutations in Sindbis virus mutants.
  • Nucleotide sequencing to identify specific genetic alterations.

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  • Complementation analysis to assess the function of viral mutants.
  • Analysis of negative-strand RNA synthesis in infected cells.

    • Main Results:

    • The mutation in Sindbis virus HR ts4 was identified as a C-to-U change at nucleotide 4903 in the nsP3 open reading frame, resulting in an Ala-268 to Val substitution.
    • Mutations in nsP3 (ts4 and ts7) selectively impaired early negative-strand synthesis, affecting the formation and/or function of the initial replication complex.
    • Neither ts4 nor ts7 complemented nsP2 mutants, suggesting nsP2 and nsP3 function as a single cistron early in infection.

    Conclusions:

    • Sindbis virus nsP3 plays a critical role in the early stages of negative-strand RNA synthesis.
    • Mutations in nsP3 disrupt the formation of the initial alphavirus replication complex.
    • nsP2 and nsP3 likely form a tightly associated complex (P23) essential for initiating viral RNA replication.