Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses

Virus Res. 2012 Aug;167(2):247-58. doi: 10.1016/j.virusres.2012.05.006. Epub 2012 May 19.

Abstract

The purpose of this study was to compare the biochemical and biological properties of nonstructural protein (nsp) 15 among mouse hepatitis virus (MHV), severe acute respiratory syndrome coronavirus (SARS-CoV) and transmissible gastroenteritis virus (TGEV) in virus-infected and ectopically expressed cells. In virus-infected cells, MHV nsp15 distributed unevenly throughout the cytoplasm but predominantly in the perinuclear region. When expressed as N-terminal enhanced green fluorescence protein (EGFP) fusion, it predominantly formed speckles in the cytoplasm. In contrast, SARS-CoV and TGEV EGFP-nsp15s distributed smoothly in the whole cell and did not form speckles. Deletion mapping experiments identified two domains responsible for the speckle formation in MHV EGFP-nsp15: Domain I (aa101-150) and Domain III (aa301-374). Interestingly, Domain II (aa151-250) had an inhibitory effect on Domain III- but not on Domain I-mediated speckle formation. Expression of a small (35aa) sequence in Domain III alone was sufficient to form speckles for all 3 viral nsp15s. However, addition of surrounding sequences in Domain III abolished the speckle formation for TGEV nsp15 but not for MHV and SARS-CoV nsp15s. Further domain swapping experiments uncovered additional speckle-inducing and -suppressive elements in nsp15s of SARS-CoV and TGEV. Homotypic interaction involving Domain III of MHV nsp15 was further demonstrated biochemically. Moreover, the biological functions of the expressed nsp15s were assessed in MHV-infected cells. It was found that the effects of EGFP-nsp15s on MHV replication were both virus species- and nsp15 domain-dependent. Collectively these results thus underscore the differential biochemical and biological functions among the nsp15s of MHV, TGEV and SARS-CoV in host cells.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cells, Cultured
  • Cytoplasm / chemistry
  • DNA Mutational Analysis
  • Humans
  • Mice
  • Murine hepatitis virus / enzymology*
  • Murine hepatitis virus / genetics
  • Protein Structure, Tertiary
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Sequence Deletion
  • Severe acute respiratory syndrome-related coronavirus / enzymology*
  • Severe acute respiratory syndrome-related coronavirus / genetics
  • Transmissible gastroenteritis virus / enzymology*
  • Transmissible gastroenteritis virus / genetics
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • Viral Nonstructural Proteins
  • RNA-Dependent RNA Polymerase