PMID:21900421

Citation	Kuchta, AL, Parikh, H, Zhu, Y, Kellogg, GE, Parris, DS and McVoy, MA (2012) Structural modelling and mutagenesis of human cytomegalovirus alkaline nuclease UL98. J. Gen. Virol. 93:130-8
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.
Links	PubMed PMC3352332 Online version:10.1099/vir.0.034876-0
Keywords	Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Catalytic Domain; Cell Line; Cytomegalovirus/chemistry; Cytomegalovirus/enzymology; Cytomegalovirus/genetics; Cytomegalovirus/physiology; Cytomegalovirus Infections/virology; Humans; Immediate-Early Proteins/chemistry; Immediate-Early Proteins/genetics; Immediate-Early Proteins/metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HCMVM:AN	enables	GO:0004520: endodeoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HCMVM:AN	enables	GO:0004529: exodeoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HCMVM:AN		GO:0004529: exodeoxyribonuclease activity	ECO:0000314:		F		Figure 2 shows the results demonstrating UL98's exonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled. They were then combined with 14C-labelled DNA. The amount of cleaved DNA was measured over time. Figure 2a shows the significantly different cleavage rates between the WT gene and the R164A mutant, whose function was greatly impaired. Figure 2b show the total cleavage done by the WT and all the mutants over the course of an hour. The WT gene product exhibited significantly more exonuclease activity than the UL98-impaired mutants.	complete CACAO 7973
HCMVM:AN		GO:0004520: endodeoxyribonuclease activity	ECO:0000314:		F		Figure 3 shows the results demonstrating UL98's endonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled. In figure 3a, the WT and mutants were incubated with closed-circular plasmid DNA to see if the gene products would convert it to open-circular and linear forms using endonuclease activity. Following incubation, gel electrophoresis measured the leftover DNA products. The WT and one of the mutants were able to degrade the entire DNA substrate by nicking the supercoiled substrate and processing the products into smaller fragments not visible on the gel. Other mutants were only able to nick the supercoiled DNA, but not digest anything else. In figure 3b, the WT and mutants were incubated with a ssDNA substrate that had a 3' quencher and a 5' fluorophore. Endonucleolytic cleavage would result in detectable fluorescence. Over time, more protein was added to the reaction in increments. The WT and one of the mutants produced significantly more fluorescence than the rest of the mutants. This indicates that those mutants suffered from impaired endonuclease function. The R164A mutant had a slightly reduced endonuclease activity compaired to the WT.	complete CACAO 7974

References

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