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

Author:
Cao, Jianzhong, Zhang, Xuming
Source:
Virus research 2012 v.167 no.2 pp. 247-258
ISSN:
0168-1702
Subject:
Murine hepatitis virus, Severe acute respiratory syndrome coronavirus, Transmissible gastroenteritis virus, chromosome mapping, cytoplasm, green fluorescent protein, mice, swine, viral nonstructural proteins, viruses
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.
Agid:
846399