Sensitivity of monkey B virus (Cercopithecine herpesvirus 1) to antiviral drugs: role of thymidine kinase in antiviral activities of substrate analogs and acyclonucleosides.

Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.

Identification and analysis of the simian varicella virus thymidine kinase gene.

The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.