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Journal of Virology, December 2005, p. 14570-14585, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14570-14585.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of Mutations in the Mouse Hepatitis Virus 3'(+)42 Protein Binding Element on RNA Replication

Reed F. Johnson ,1,{dagger},{ddagger} Min Feng,1,{dagger} Pinghua Liu,1 Jason J. Millership,1,§ Boyd Yount,2 Ralph S. Baric,2 and Julian L. Leibowitz1*

Department of Pathology and Laboratory Medicine, Texas A&M University System College of Medicine, College Station, Texas 77843-1114,1 Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-74352

Received 18 October 2004/ Accepted 13 July 2005

The mouse hepatitis virus (MHV) genome's 3' untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3'-terminal 42 nucleotides [3'(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3'(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3'-terminal 42-nucleotide element. Additionally we have identified a role for the 3'-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3'(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3' end of the MHV genome in subgenomic RNA synthesis.

* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Texas A&M University System College of Medicine, 208 Reynolds Medical Building, 1114 TAMU, College Station, TX 77843-1114. Phone: (979) 845-7288. Fax: (979) 862-1299. E-mail: jleibowitz{at}tamu.edu.

{ddagger} Present address: Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104-6049.

§ Present address: Fort Dodge Animal Health, 800 5th St. NW, P.O. Box 518, Fort Dodge, IA 50501.

{dagger} These two authors contributed equally to this work.

Journal of Virology, December 2005, p. 14570-14585, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14570-14585.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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