Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation.
The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of ICP10 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 degrees C compared to 34 degrees C after 12 h post-infection. The efficiency of plaquing is 0.003. The growth defect at 39 degrees C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP10 gene from ts5-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 degrees C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 degrees C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors pJW17 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.
Smith
C C
CC
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Kulka
M
M
Wymer
J P
JP
Chung
T D
TD
Aurelian
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004795:Enzyme Stability; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000096:biosynthesis*; D016415:Sequence Alignment; D018139:Simplexvirus / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D013696:Temperature; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis
D014764:Viral Proteins; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D004259:DNA-Directed DNA Polymerase