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 59 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.

Mitochondrial Nucleases ENDOG and EXOG Participate in Mitochondrial DNA Depletion Initiated by Herpes Simplex Virus 1 UL12.5

“…Both mutations abolish the ability of UL12 to complement the growth defect of a UL12-null mutant virus (29). Mutagenesis of EBV, HCMV, and KSHV UL12 orthologs has also demonstrated that residues analogous to UL12 G336, S338, and D340 are critical for nuclease activity (30,(32)(33)(34). We included three additional mutations that have also been shown to eliminate detectable nuclease activity in vitro and abolish complementing activity in vivo; ⌬N, L150K, and ⌬C (23).…”

Anti-cytomegalovirus activity of the anthraquinone atanyl blue PRL

“…Recombinant UL98 was inducibly expressed in Escherichia coli BL21 (DE3) lys S and purified using methods modified from those previously described ( Kuchta et al, 2012 ). The UL98 expression vector pD17–98(wt) ( Kuchta et al, 2012 ) was modified to contain an N-terminal sequence encoding two tandem histidine (his 6 ) tags.…”

“…Impaired replication has been linked to defects in DNA processing, capsid stability, and capsid nuclear egress ( Martinez et al, 1996 , Porter and Stow, 2004 , Shao et al, 1993 , Weller et al, 1990 ). Recently our group showed that a CMV UL98 null mutant is severely compromised for replication ( Kuchta et al, 2012 ). At least one function of the CMV UL98 is likely to be similar to HSV-1 UL12 since the CMV UL98 gene can functionally complement the replication defect of HSV-1 UL12 null viruses ( Gao et al, 1998 ).…”

Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

“…3C). Proximity of the T172P mutation to the reported 5=-phosphate-binding pocket and catalytic active site in other viral nucleases likely prevents proper substrate binding, cleavage, and release, possibly due to proline-dependent protein misfolding (21,22).To further characterize VZV ORF48 protein nuclease activity, 500 ng undigested (circular) pCI-Neo DNA was examined for endonuclease activity. Undigested plasmid DNA consisted of both nicked (relaxed circular, RC) and supercoiled (SC) DNA (Fig.…”

“…3C). Proximity of the T172P mutation to the reported 5=-phosphate-binding pocket and catalytic active site in other viral nucleases likely prevents proper substrate binding, cleavage, and release, possibly due to proline-dependent protein misfolding (21,22).…”