Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket.
The rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly.
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8026, USA.
Kristen M
KM
Ogden
Harish N
HN
Ramanathan
John T
JT
Patton
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism*; D012089:Mammalian orthoreovirus 3 / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012401:Rotavirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D009711:Nucleotides; C069187:VP1 protein, Rotavirus; D014758:Viral Core Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase