Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus.
The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities. The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain. Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed. Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities. Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity. Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity. However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used. Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.
Schnierle
B S
BS
Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
Gershon
P D
PD
Moss
B
B
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004252:DNA Mutational Analysis; D005982:Glutathione Transferase / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009708:Nucleotidases / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D008780:Methyltransferases; C032117:cap I RNA (nucleoside-2'-)methyltransferase; C032116:cap II RNA(nucleoside-2'-)methyltransferase; D005982:Glutathione Transferase; D011062:Polynucleotide Adenylyltransferase; D009708:Nucleotidases