Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

ABSTRACT IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

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Inhibition of Varicella-Zoster Virus by Penciclovir in Cell Culture and Mechanism of Action

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029600700203 ◽

1996 ◽

Vol 7 (2) ◽

pp. 71-78 ◽

Cited By ~ 13

Author(s):

T. H. Bacon ◽

J. Gilbart ◽

B. A. Howard ◽

R. Standring-Cox

Keyword(s):

Dna Synthesis ◽

Varicella Zoster Virus ◽

Clinical Isolates ◽

Continuous Treatment ◽

Lung Fibroblasts ◽

Human Embryonic Lung ◽

Viral Dna ◽

Plaque Reduction Assay ◽

Varicella Zoster ◽

Infected Cells

The effect of penciclovir (BRL 39123) on the replication of varicella-zoster virus (VZV) in human embryonic lung fibroblasts (MRC-5 cells) was similar to aciclovir when the compounds were present continuously. However, when the compounds were withdrawn the antiviral activity of penciclovir was maintained more effectively than that of aciclovir. In the plaque reduction assay, median 50% effective concentrations (EC50s) were 3.8 μg ml−1 for penciclovir and 4.2 μg ml−1 for aciclovir ( n = 29 clinical isolates). Similarly, penciclovir and aciclovir were equally effective in reducing the numbers of VZV-infected MRC-5 cells and in reducing VZV DNA synthesis within infected cells following continuous treatment. Within VZV-infected cells (S)-penciclovir-triphosphate was formed from penciclovir with >95% enantiomeric purity, and the concentration of penciclovir-triphosphate was 360-fold greater than aciclovir-triphosphate immediately after treatment. This phosphorylation ratio compensates for the lower affinity of VZV DNA polymerase for penciclovir-triphosphate compared with aciclovir-triphosphate (Kis = 7.5 μM and 0.2 μM, respectively). When VZV-infected cultures were treated for 3 days, followed by withdrawal of the compound, inhibition of viral DNA synthesis by penciclovir was maintained for 24 h, whereas viral DNA synthesis resumed more readily after removal of aciclovir. Furthermore, following 8 h daily pulse treatment for 5 days, penciclovir was significantly more active than aciclovir in reducing VZV DNA synthesis ( p = 0.006, n = 10 clinical isolates). The long intracellular half-life of penciclovir-triphosphate (9.1 h) compared with that of aciclovir-triphosphate (0.8 h) accounts for the sustained inhibition of virus replication by penciclovir. This property may contribute to the clinical efficacy of famciclovir, the oral form of penciclovir.

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