Rotavirus Open Cores Catalyze 5'-capping and Methylation of Exogenous RNA: Evidence That VP3 Is a Methyltransferase

Virology. 1999 Dec 5;265(1):120-30. doi: 10.1006/viro.1999.0029.

Abstract

Rotavirus open cores prepared from purified virions consist of three proteins: the RNA-dependent RNA polymerase, VP1; the core shell protein, VP2; and the guanylyltransferase, VP3. In addition to RNA polymerase activity, open cores have been shown to contain a nonspecific guanylyltransferase activity that caps viral and nonviral RNAs in vitro. In this study, we examined the structure of RNA caps made by open cores and have analyzed open cores for other capping-related enzymatic activities. Utilizing RNase digestion and thin-layer chromatography, we found that the majority ( approximately 70%) of caps made by open cores contain the tetraphosphate linkage, GppppG, rather than the triphosphate linkage, GpppG, found on mRNAs made by rotavirus double-layered particles. Enzymatic analysis indicated that the GppppG caps resulted from the lack of a functional RNA 5'-triphosphatase in open cores, to remove the gamma-phosphate from the RNA prior to capping. RNA 5'-triphosphatases commonly exhibit an associated nucleoside triphosphatase activity, and this too was not detected in open cores. Caps of some RNAs contained an extra GMP moiety (underlined) and had the structure 3'-GpGp(p)ppGpGpC-RNA-3'. The origin of the extra GMP is not known but may reflect the cap serving as a primer for RNA synthesis. Methylated caps were produced in the presence of the substrate, S-adenosyl-l-methionine (SAM), indicating that open cores contain methyltransferase activity. UV cross-linking showed that VP3 specifically binds SAM. Combined with the results of earlier studies, our results suggest that the viral guanylyltransferase and methyltransferase are both components of VP3 and, therefore, that VP3 is a multifunctional capping enzyme.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acid Anhydride Hydrolases / metabolism
  • Catalysis
  • DNA-Directed RNA Polymerases / metabolism
  • Dinucleoside Phosphates / metabolism
  • Guanosine Triphosphate / metabolism
  • Methylation
  • Methyltransferases / metabolism*
  • Molecular Weight
  • RNA Caps / metabolism*
  • RNA, Viral / metabolism*
  • Rotavirus / metabolism*
  • S-Adenosylmethionine / metabolism
  • Single-Strand Specific DNA and RNA Endonucleases / metabolism
  • Ultraviolet Rays
  • Uridine Triphosphate / metabolism
  • Viral Proteins

Substances

  • Dinucleoside Phosphates
  • RNA Caps
  • RNA, Viral
  • Viral Proteins
  • 7-methyl-diguanosine triphosphate
  • S-Adenosylmethionine
  • Guanosine Triphosphate
  • Methyltransferases
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Single-Strand Specific DNA and RNA Endonucleases
  • Acid Anhydride Hydrolases
  • RNA triphosphatase
  • Uridine Triphosphate