VZV Foundation - A Decade Of Leadership In The Fight Against VZV Infections

	Oral Presentation Abstracts: 32 [32] PHOSPHORYLATION OF VZV REGULATORY AND STRUCTURAL PROTEINS BY A CLONED, BIOLOGICALLY ACTIVE ORF47 PROTEIN KINASE *T.K. Kenyon (1), J. Lynch (2), J. Hay (2), W. Ruyechan (2), and C. Grose (1). (1) Departments of Microbiology and Pediatrics, University of Iowa, Iowa City, Iowa, USA; (2) Department of Microbiology and Markey Center for Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, Buffalo, New York, USA. Varicella-Zoster Virus encodes two genes, ORF47 and ORF66, with amino acid motifs similar to classical kinase domains. Neither ORF47 nor the HSV homolog UL13 had been active in vitro when purified from transfected cells, and thus the possibility remained that the phosphorylation attributed to them was due to the viral protein regulating another viral or cellular kinase. Preservation of the intrinsic kinase activity of recombinant, epitope-tagged ORF47 immunoprecipitated from transfected cells requires stringent conditions, including careful regulation of pH, pretreatment with phosphatase inhibitors and cytoskeleton disrupters, reduction of detergents, presence of polyamines, and inclusion of protease inhibitors, phosphatase inhibitors, specific ions, and heparin. Data were analyzed by a Hewett-Packard InstantImager. In this in vitro kinase assay, ORF47 phosphorylated VZV regulatory proteins ORF62 (abundant in the tegument and during primary infection) and ORF63 (abundant during VZV reactivation). When regions with extensive acidic clusters around a serine or threonine (AC) were truncated in mutants, phosphorylation was abolished. ORF47 bound tightly to these proteins such that the kinase co-precipitated the full-length regulatory proteins from the kinase reactions even after extensive washing. In addition, the cloned ORF47 phosphorylated the viral glycoprotein gE, which also has an extensive acidic motif (AC). ORF47 did not phosphorylate a VZV gE with a truncated carboxy domain that lacked the AC, or VZV gB, which does not encode an AC but can be phosphorylated by CKII. The kinase and full-length gE coprecipitated from infected cells, and ORF47 was a functional component of the VZV Fc receptor complex. This experiment firmly established that gE phosphorylation was due to ORF47 and not due to contaminating CKII. In summary, these experiments demonstrated that the ORF47 phosphorylation consensus sequence was more stringent than the CKII consensus sequence and required an AC, a highly acidic cluster around the phosphorylated residue. Corresponding Author: T.K. Kenyon, Department of Microbiology, University of Iowa, 1612 SB, Iowa City, IA 52242, USA

Oral Presentation Abstracts: 32

[32]

PHOSPHORYLATION OF VZV REGULATORY AND STRUCTURAL PROTEINS BY A CLONED, BIOLOGICALLY ACTIVE ORF47 PROTEIN KINASE

*T.K. Kenyon (1), J. Lynch (2), J. Hay (2), W. Ruyechan (2), and C. Grose (1).
(1) Departments of Microbiology and Pediatrics, University of Iowa, Iowa City, Iowa, USA; (2) Department of Microbiology and Markey Center for Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, Buffalo, New York, USA.

Varicella-Zoster Virus encodes two genes, ORF47 and ORF66, with amino acid motifs similar to classical kinase domains. Neither ORF47 nor the HSV homolog UL13 had been active in vitro when purified from transfected cells, and thus the possibility remained that the phosphorylation attributed to them was due to the viral protein regulating another viral or cellular kinase.
Preservation of the intrinsic kinase activity of recombinant, epitope-tagged ORF47 immunoprecipitated from transfected cells requires stringent conditions, including careful regulation of pH, pretreatment with phosphatase inhibitors and cytoskeleton disrupters, reduction of detergents, presence of polyamines, and inclusion of protease inhibitors, phosphatase inhibitors, specific ions, and heparin. Data were analyzed by a Hewett-Packard InstantImager.
In this in vitro kinase assay, ORF47 phosphorylated VZV regulatory proteins ORF62 (abundant in the tegument and during primary infection) and ORF63 (abundant during VZV reactivation). When regions with extensive acidic clusters around a serine or threonine (AC) were truncated in mutants, phosphorylation was abolished. ORF47 bound tightly to these proteins such that the kinase co-precipitated the full-length regulatory proteins from the kinase reactions even after extensive washing.
In addition, the cloned ORF47 phosphorylated the viral glycoprotein gE, which also has an extensive acidic motif (AC). ORF47 did not phosphorylate a VZV gE with a truncated carboxy domain that lacked the AC, or VZV gB, which does not encode an AC but can be phosphorylated by CKII. The kinase and full-length gE coprecipitated from infected cells, and ORF47 was a functional component of the VZV Fc receptor complex. This experiment firmly established that gE phosphorylation was due to ORF47 and not due to contaminating CKII.
In summary, these experiments demonstrated that the ORF47 phosphorylation consensus sequence was more stringent than the CKII consensus sequence and required an AC, a highly acidic cluster around the phosphorylated residue.

Corresponding Author: T.K. Kenyon, Department of Microbiology, University of Iowa, 1612 SB, Iowa City, IA 52242, USA