Construction and characterization of herpes simplex virus type 2 mutants deleted in the protein kinase or ribonucleotide reductase domains of the chimeric ICP10 protein

Abstract

The large subunit of ribonucleotide reductase (RR1) encoded by Herpes Simplex Virus Type 1 and 2 (ICP6 and ICP10, respectively) is a chimeric protein consisting of a Ser/Threonine protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase (RR) domain at the carboxy terminus. The findings that the PK domain is present only in HSV RR1 proteins, it is dispensable for ribonucleotide reduction and it functions as immediate-early (IE) protein during HSV infection, suggest that the PK activity plays a significant role in virus pathogenesis. The present work was initiated to confirm the intrinsic nature of ICP10 PK activity and to elucidate its role in HSV-2 infection. In stably transfected eukaryotic cells, ICP10 PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the transmembrane (TM) segment and it was significantly impaired by mutation of the invariant Lys (Lys{dollar}\sp{lcub}176{rcub}{dollar}). Loss of PK activity by Lys{dollar}\sp{lcub}176{rcub}{dollar} mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29{dollar}\rm\sp{lcub}la1{rcub}{dollar}) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. In cells infected with laboratory and patient isolates of HSV, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and Immunoglobulin G (IgG). Two HSV-2 (G) mutants deleted in the protein kinase or ribonucleotide reductase domains of ICP10 (ICP10{dollar}\Delta{dollar}PK and ICP10{dollar}\Delta{dollar}RR, respectively) were constructed by marker transfer. ICP10{dollar}\Delta{dollar}PK virus lost its intrinsic PK activity but retained its RR activity; ICP10{dollar}\Delta{dollar}RR virus retained its PK activity but lost its RR activity. ICP10{dollar}\Delta{dollar}PK virus does not replicate during the first 10 hrs postinfection (p.i.). However, its titers catch up with those of the wild type virus by 24 hrs p.i.; ICP10{dollar}\Delta{dollar}RR virus replicates as well as the wild type virus in exponentially growing cells but it is significantly impaired for growth in growth-restricted cells. HSV-2 and ICP10{dollar}\Delta{dollar}RR virus produce similar clear plaques but ICP10{dollar}\Delta{dollar}PK virus produces hazy plaques, which under magnification consist of a mixture of lysed and unlysed cells. The studies suggest that (i) ICP10 has intrinsic auto- and transphosphorylating PK activity, (ii) ICP10 PK and RR are functionally dissociable in virus infected cells, (iii) ICP10 PK is required for virus replication during the first 10 hrs p.i. and (iv) ICP10 PK may be involved in cell death in virus infected cells.