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Journal of Virology, December 2009, p. 12084-12093, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00915-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mouse Hepatitis Virus Stem-Loop 2 Adopts a uYNMG(U)a-Like Tetraloop Structure That Is Highly Functionally Tolerant of Base Substitutions{triangledown}

Pinghua Liu,1 Lichun Li,2,{dagger} Sarah C. Keane,3 Dong Yang,1 Julian L. Leibowitz,1* and David P. Giedroc2,3*

Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center College of Medicine, College Station, Texas 77843-1114,1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128,2 Department of Chemistry, Indiana University, Bloomington, Indiana 474053

Received 7 May 2009/ Accepted 3 September 2009

Stem-loop 2 (SL2) of the 5'-untranslated region of the mouse hepatitis virus (MHV) contains a highly conserved pentaloop (C47-U48-U49-G50-U51) stacked on a 5-bp stem. Solution nuclear magnetic resonance experiments are consistent with a 5'-uYNMG(U)a or uCUYG(U)a tetraloop conformation characterized by an anti-C47-syn-G50 base-pairing interaction, with U51 flipped out into solution and G50 stacked on A52. Previous studies showed that U48C and U48A substitutions in MHV SL2 were lethal, while a U48G substitution was viable. Here, we characterize viruses harboring all remaining single-nucleotide substitutions in the pentaloop of MHV SL2 and also investigate the degree to which the sequence context of key pentaloop point mutations influences the MHV replication phenotype. U49 or U51 substitution mutants all are viable; C47 substitution mutants also are viable but produce slightly smaller plaques than wild-type virus. In contrast, G50A and G50C viruses are severely crippled and form much smaller plaques. Virus could not be recovered from G50U-containing mutants; rather, only true wild-type revertants or a virus, G50U/C47A, containing a second site mutation were recovered. These functional data suggest that the Watson-Crick edges of C47 and G50 (or A47 and U50 in the G50U/C47A mutant) are in close enough proximity to a hydrogen bond with U51 flipped out of the hairpin. Remarkably, increasing the helical stem stability rescues the previously lethal mutants U48C and G50U. These studies suggest that SL2 functions as an important, but rather plastic, structural element in stimulating subgenomic RNA synthesis in coronaviruses.

* Corresponding authors. Mailing address for J. Leibowitz: Texas A&M Health Science Center, 208 Joe H. Reynolds Medical Bldg., 114 TAMU, College Station, TX 77843-1114. Phone: (979) 845-7288. Fax: (979) 845-3479. E-mail: jleibowitz{at}tamu.edu. Mailing address for D. P. Giedroc: Department of Chemistry, Indiana University, 212 S. Hawthorne Drive, Bloomington, IN 47405. Phone: (812) 856-3178. Fax: (812) 856-5710. E-mail: giedroc{at}indiana.edu

{triangledown} Published ahead of print on 16 September 2009.

{dagger} Present address: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405.

Journal of Virology, December 2009, p. 12084-12093, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00915-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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