A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Virology. 2015 Oct;484:313-322. doi: 10.1016/j.virol.2015.06.016. Epub 2015 Jul 3.

Abstract

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

Keywords: G-quadruplex; MERS-CoV; Macrodomain; Reverse genetics; SARS-CoV replicon; SARS-unique domain; X-domain.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Genes, Reporter
  • Luciferases, Renilla / analysis
  • Luciferases, Renilla / genetics
  • Protein Structure, Tertiary
  • Reverse Genetics
  • SARS Virus / genetics
  • SARS Virus / physiology*
  • Sequence Deletion
  • Staining and Labeling
  • Transcription, Genetic*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virus Replication*

Substances

  • Viral Nonstructural Proteins
  • Luciferases, Renilla