Expression of the HSV-2 ribonucleotide reductase subunits in adenovirus vectors or stably transformed cells: restoration of enzymatic activity by reassociation of enzyme subunits in the absence of other HSV proteins.

Abstract

We have cloned the large subunit (RR1) of the HSV-2 ribonucleotide reductase into a helper-independent adenovirus 5 vector under control of the viral major late promoter. Infection of 293 cells with the AdRed-1 recombinant virus resulted in the expression of the HSV-2 RR1 protein. We have also produced cells which constitutively express the small (RR2) subunit of the HSV-2 enzyme by transfecting 293 cells with a plasmid encoding this protein and the neo resistance marker (pSV2neo-RR2). Infection of the A439-14 producer cells with AdRed-1 resulted in the expression of enzymatically active HSV-2 ribonucleotide reductase. HSV-2 reductase activity could also be detected upon mixing of extracts from cells expressing either subunit. Our results indicate that the HSV-2 holoenzyme can be reconstituted in vivo and in vitro and that no HSV-2 proteins, beyond the enzyme subunits, are required for the formation and activity of the viral reductase.