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Journal of Virology, June 2009, p. 6087-6097, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.00160-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements{triangledown}

Kortney M. Gustin,1 Bo-Jhih Guan,1 Agnieszka Dziduszko,2 and David A. Brian1,2*

Departments of Microbiology,1 Pathobiology, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996-08452

Received 23 January 2009/ Accepted 29 March 2009

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 ~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 ~2.5 µM 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.

* Corresponding author. Mailing address: Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845. Phone: (865) 974-4030. Fax: (865) 974-4007. E-mail: dbrian{at}utk.edu

{triangledown} Published ahead of print on 8 April 2009.

Journal of Virology, June 2009, p. 6087-6097, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.00160-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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