|dc.description.abstract||Nidoviruses are a monophyletic group of RNA viruses with exceptionally large genomes ranging from 12 - 41 kb. Despite their widely varying genome sizes, members of the order Nidovirales share common characteristic features, such as an array of conserved domains in their replicase genes and the expression of structural and accessory proteins from a nested set of sub-genomic mRNAs. The replicase gene occupies the 5'-terminal two-thirds of the genome and encodes for nonstructural proteins (nsp) that form active replication-transcription complexes (RTC) and have other important functions in the viral replication cycle. Previously, attempts to develop in vitro polymerase assays for coronaviruses and a few other nidoviruses resulted in contradictory results. Reverse genetics and biochemical studies have suggested that, in coronaviruses, nsp7, nsp8 and nsp12 are the key factors required for RNA synthesis in vitro. In this work, recombinant and tag-less forms of nsp7, nsp8 and the pre-assembled nsp(7+8) complex were produced and used to investigate the polymerase activity ofnsp12, a two-domain protein harboring the canonical RNA-dependent RNA polymerase (RdRp) domain in its C-terminal region. The studies revealed that a pre-assembled coronavirus nsp(7+8)+nsp12 complex was required for efficient elongation activityfrom partially double-stranded (‛primed’) RNA substrates in the presence of Mg2+ or Mn2+.
In the absence of nsp12, human coronavirus 229E (HCoV-229E) nsp8 and a pre assembled nsp(7+8) complex exhibited RNA 3'-terminal adenylyltransferase activity on different RNA substrates but failed to produce complementary copies of heteropolymeric template RNAs. Production and characterization of nsp7/8/12 complexes of representative alpha- and betacoronaviruses revealed that all these complexes were able to initiate RNA synthesis de novo from single-stranded template RNAs in the presence of Mn2+ but not Mg2+, suggesting that coronavirus replicase complexes do not strictly depend on the presence of (RNA or protein) primers to initiate RNA synthesis, at least in vitro. With very few exceptions, RdRp activity of nsp12 was revealed to require the presence of the cognate nsp(7+8) complex, while nsp(7+8) complexes from other coronavirus species failed to attain RdRp activity, suggesting important functional and/or structural constraints in the formation of active nsp7/8/12 complexes. Amino acid substitutions in the conserved RdRp motifs A and C, respectively, abolished both de novo and primer-dependent activity. At physiological Mg2+ or Mn2+ concentrations, the HCoV-229E nsp(7+8)+nsp12 complex was shown to differentiate between rNTPs and dNTPs and to incorporate the correct nucleotides during RNA elongation. Further investigations using a range of alpha- and betacoronavirus nsp(7+8) complexes produced in E. coli provided evidence that feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV)nsp(7+8) predominantly form heterotrimeric complexes (2:1), while HCoV-229E, PEDV, SARS-CoV and MERS-CoV produce predominantly heterotetrameric nsp(7+8) complexes (2:2). In all these cases, nsp(7+8) heterodimeric (1:1) complexes were detected, suggesting that they form the building block for larger complexes in all coronaviruses.
In another part of the study, the arterivirus equine arteritis virus (EAV) nsp9 (harboring the RdRp domain) was characterized. Although the present study, in line with previous studies, failed to provide conclusive evidence for nsp9-mediated RdRp activity, the study showed that the protein has RNA 3'-terminal adenylyltransferase activity and employs its N-terminal NiRAN domain to transfer UMP and GMP, respectively, to the N-terminus of the arteriviral nsp7 or nsp7α. Mass spectrometry analyses confirmed that nsp9 mediates the transfer of one molecule of NMP to the acceptor protein.||de_DE