title: Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses.
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.
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Cao
authors.forename: Xuming
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D004252:DNA Mutational Analysis; D006801:Humans; D051379:Mice; D006517:Murine hepatitis virus / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D005760:Transmissible gastroenteritis virus / Q000201:enzymology* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase