title: Structural modelling and mutagenesis of human cytomegalovirus alkaline nuclease UL98.
abstract: Human cytomegalovirus encodes an alkaline nuclease, UL98, that is highly conserved among herpesviruses and has both endonuclease (endo) and exonuclease (exo) activities. This protein is thought to be important for viral replication and therefore represents a potential target for antiviral development; however, little is known about its structure or role in viral replication. Comparative structural modelling was used to build a model of UL98 based on the known structure of shutoff and exonuclease protein from Kaposi's sarcoma-associated herpesvirus. The model predicts that UL98 residues D254, E278 and K280 represent the critical aspartic acid, glutamic acid and lysine active-site residues, respectively, while R164 and S252 correspond to residues proposed to bind the 5' phosphate of the DNA substrate. UL98 with an amino-terminal hexahistidine tag was expressed in Escherichia coli, purified by affinity chromatography and confirmed to have exo and endo activities. Amino acid substitutions D254A, E278A, K280A and S252A virtually eliminated exo and endo activities, whereas R164A retained full endo activity but only 10 % of the exo activity compared with the wild-type enzyme. A mutant virus lacking UL98 was viable but severely attenuated for replication, while one expressing UL98(R164A) replicated normally. These results confirm the utility of the model in representing the active-site region of UL98 and suggest a mechanism for the differentiation of endonuclease and exonuclease activities. These findings could facilitate the exploration of the roles of alkaline nucleases in herpesvirus replication and the rational design of inhibitors that target their enzymic activities.
authors: Department of Pediatrics, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors: Alison L
authors: AL
authors: Kuchta
authors: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298-0540, USA.
authors: Hardik
authors: H
authors: Parikh
authors: Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.
authors: Yali
authors: Y
authors: Zhu
authors: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298-0540, USA.
authors: Glen E
authors: GE
authors: Kellogg
authors: Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.
authors: Deborah S
authors: DS
authors: Parris
authors: Department of Pediatrics, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors: Michael A
authors: MA
authors: McVoy
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D003587:Cytomegalovirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000821:virology; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014779:Virus Replication
chemical_list: D017874:Immediate-Early Proteins; C095110:UL98 protein, cytomegalovirus