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Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements

J Virol. 2009 Jun;83(12):6087-97. doi: 10.1128/JVI.00160-09. Epub 2009 Apr 8.

Abstract

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

Publication types

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

MeSH terms

  • 3' Untranslated Regions / genetics
  • 5' Untranslated Regions / genetics
  • Base Sequence
  • Cell Line, Tumor
  • Coronavirus, Bovine / genetics*
  • Coronavirus, Bovine / physiology
  • Gene Expression Regulation, Viral
  • Humans
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Protein Binding
  • RNA, Viral / biosynthesis
  • RNA, Viral / genetics
  • RNA-Binding Proteins / genetics*
  • Sequence Analysis, RNA
  • Substrate Specificity
  • Viral Nonstructural Proteins / genetics*
  • Virus Replication*

Substances

  • 3' Untranslated Regions
  • 5' Untranslated Regions
  • RNA, Viral
  • RNA-Binding Proteins
  • Viral Nonstructural Proteins
  • nonstructural protein, coronavirus