1887

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 , 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.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.034876-0
2012-01-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/1/130.html?itemId=/content/journal/jgv/10.1099/vir.0.034876-0&mimeType=html&fmt=ahah

References

  1. Adam B. L., Jervey T. Y., Kohler C. P., Wright G. L. Jr, Nelson J. A., Stenberg R. M.. ( 1995;). The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein. . J Virol 69:, 5304–5310.[PubMed]
    [Google Scholar]
  2. Bagnéris C., Briggs L. C., Savva R., Ebrahimi B., Barrett T. E.. ( 2011;). Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff. . Nucleic Acids Res 39:, 5744–5756. [CrossRef][PubMed]
    [Google Scholar]
  3. Balasubramanian N., Bai P., Buchek G., Korza G., Weller S. K.. ( 2010;). Physical interaction between the herpes simplex virus type 1 exonuclease, UL12, and the DNA double-strand break-sensing MRN complex. . J Virol 84:, 12504–12514. [CrossRef][PubMed]
    [Google Scholar]
  4. Buisson M., Géoui T., Flot D., Tarbouriech N., Ressing M. E., Wiertz E. J., Burmeister W. P.. ( 2009;). A bridge crosses the active-site canyon of the Epstein–Barr virus nuclease with DNase and RNase activities. . J Mol Biol 391:, 717–728. [CrossRef][PubMed]
    [Google Scholar]
  5. Bujnicki J. M., Rychlewski L.. ( 2001;). The herpesvirus alkaline exonuclease belongs to the restriction endonuclease PD-(D/E)XK superfamily: insight from molecular modeling and phylogenetic analysis. . Virus Genes 22:, 219–230. [CrossRef][PubMed]
    [Google Scholar]
  6. Cui X., McGregor A., Schleiss M. R., McVoy M. A.. ( 2008;). Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication. . J Virol Methods 149:, 231–239. [CrossRef][PubMed]
    [Google Scholar]
  7. Dahlroth S. L., Gurmu D., Schmitzberger F., Engman H., Haas J., Erlandsen H., Nordlund P.. ( 2009;). Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi’s sarcoma-associated herpesvirus. . FEBS J 276:, 6636–6645. [CrossRef][PubMed]
    [Google Scholar]
  8. Dunn W., Chou C., Li H., Hai R., Patterson D., Stolc V., Zhu H., Liu F.. ( 2003;). Functional profiling of a human cytomegalovirus genome. . Proc Natl Acad Sci U S A 100:, 14223–14228. [CrossRef][PubMed]
    [Google Scholar]
  9. Fouda N., Caracatsanis M., Kut I. A., Hammarström L.. ( 1991;). Mineralization disturbances of the developing rat molar induced by mono- and bisphosphonates. . J Biol Buccale 19:, 106–115.[PubMed]
    [Google Scholar]
  10. Gao M., Robertson B. J., McCann P. J., O’Boyle D. R., Weller S. K., Newcomb W. W., Brown J. C., Weinheimer S. P.. ( 1998;). Functional conservations of the alkaline nuclease of herpes simplex type 1 and human cytomegalovirus. . Virology 249:, 460–470. [CrossRef][PubMed]
    [Google Scholar]
  11. Goldstein J. N., Weller S. K.. ( 1998;). The exonuclease activity of HSV-1 UL12 is required for in vivo function. . Virology 244:, 442–457. [CrossRef][PubMed]
    [Google Scholar]
  12. Henderson G. I., Hu Z. Q., Yang Y., Perez T. B., Devi B. G., Frosto T. A., Schenker S.. ( 1993;). Ganciclovir transfer by human placenta and its effects on rat fetal cells. . Am J Med Sci 306:, 151–156. [CrossRef][PubMed]
    [Google Scholar]
  13. Henderson J. O., Ball-Goodrich L. J., Parris D. S.. ( 1998;). Structure–function analysis of the herpes simplex virus type 1 UL12 gene: correlation of deoxyribonuclease activity in vitro with replication function. . Virology 243:, 247–259. [CrossRef][PubMed]
    [Google Scholar]
  14. Hoffmann P. J., Cheng Y. C.. ( 1978;). The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme. . J Biol Chem 253:, 3557–3562.[PubMed]
    [Google Scholar]
  15. Humphrey W., Dalke A., Schulten K.. ( 1996;). vmd: visual molecular dynamics. . J Mol Graph 14:, 33–38, 27–28. [CrossRef][PubMed]
    [Google Scholar]
  16. Kehm E., Göksu M., Bayer S., Knopf C. W.. ( 1998;). Herpes simplex virus type 1 DNase: functional analysis of the enzyme expressed by recombinant baculovirus. . Intervirology 41:, 110–119. [CrossRef][PubMed]
    [Google Scholar]
  17. Klug S., Lewandowski C., Merker H. J., Stahlmann R., Wildi L., Neubert D.. ( 1991;). In vitro and in vivo studies on the prenatal toxicity of five virustatic nucleoside analogues in comparison to aciclovir. . Arch Toxicol 65:, 283–291. [CrossRef][PubMed]
    [Google Scholar]
  18. Kovall R. A., Matthews B. W.. ( 1998;). Structural, functional, and evolutionary relationships between λ-exonuclease and the type II restriction endonucleases. . Proc Natl Acad Sci U S A 95:, 7893–7897. [CrossRef][PubMed]
    [Google Scholar]
  19. Liu M. T., Hu H. P., Hsu T. Y., Chen J. Y.. ( 2003;). Site-directed mutagenesis in a conserved motif of Epstein–Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases. . J Gen Virol 84:, 677–686. [CrossRef][PubMed]
    [Google Scholar]
  20. Martinez R., Shao L., Bronstein J. C., Weber P. C., Weller S. K.. ( 1996;). The product of a 1.9-kb mRNA which overlaps the HSV-1 alkaline nuclease gene (UL12) cannot relieve the growth defects of a null mutant. . Virology 215:, 152–164. [CrossRef][PubMed]
    [Google Scholar]
  21. Martinez R., Goldstein J. N., Weller S. K.. ( 2002;). The product of the UL12.5 gene of herpes simplex virus type 1 is not essential for lytic viral growth and is not specifically associated with capsids. . Virology 298:, 248–257. [CrossRef][PubMed]
    [Google Scholar]
  22. Patel A. H., Subak-Sharpe J. H., Stow N. D., Marsden H. S., Maclean J. B., Dargan D. J.. ( 1996;). Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system. . J Gen Virol 77:, 199–209. [CrossRef][PubMed]
    [Google Scholar]
  23. Porter I. M., Stow N. D.. ( 2004a;). Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12. . J Gen Virol 85:, 3501–3510. [CrossRef][PubMed]
    [Google Scholar]
  24. Porter I. M., Stow N. D.. ( 2004b;). Virus particles produced by the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12 contain abnormal genomes. . J Gen Virol 85:, 583–591. [CrossRef][PubMed]
    [Google Scholar]
  25. Reuven N. B., Staire A. E., Myers R. S., Weller S. K.. ( 2003;). The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro. . J Virol 77:, 7425–7433. [CrossRef][PubMed]
    [Google Scholar]
  26. Reuven N. B., Willcox S., Griffith J. D., Weller S. K.. ( 2004;). Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease. . J Mol Biol 342:, 57–71. [CrossRef][PubMed]
    [Google Scholar]
  27. Saccoccio F. M., Sauer A. L., Cui X., Armstrong A. E., Habib S. E., Johnson D. C., Ryckman B. J., Klingelhutz A. J., Adler S. P., McVoy M. A.. ( 2011;). Peptides from cytomegalovirus UL130 and UL131 proteins induce high titer antibodies that block viral entry into mucosal epithelial cells. . Vaccine 29:, 2705–2711. [CrossRef][PubMed]
    [Google Scholar]
  28. Sauer A., Wang J. B., Hahn G., McVoy M. A.. ( 2010;). A human cytomegalovirus deleted of internal repeats replicates with near wild type efficiency but fails to undergo genome isomerization. . Virology 401:, 90–95. [CrossRef][PubMed]
    [Google Scholar]
  29. Schleiss M. R.. ( 2005;). Antiviral therapy of congenital cytomegalovirus infection. . Semin Pediatr Infect Dis 16:, 50–59. [CrossRef][PubMed]
    [Google Scholar]
  30. Shao L., Rapp L. M., Weller S. K.. ( 1993;). Herpes simplex virus 1 alkaline nuclease is required for efficient egress of capsids from the nucleus. . Virology 196:, 146–162. [CrossRef][PubMed]
    [Google Scholar]
  31. Sheaffer A. K., Weinheimer S. P., Tenney D. J.. ( 1997;). The human cytomegalovirus UL98 gene encodes the conserved herpesvirus alkaline nuclease. . J Gen Virol 78:, 2953–2961.[PubMed]
    [Google Scholar]
  32. Silva M. C., Yu Q. C., Enquist L., Shenk T.. ( 2003;). Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. . J Virol 77:, 10594–10605. [CrossRef][PubMed]
    [Google Scholar]
  33. Stone J.. ( 1998;). An efficient library for parallel ray tracing and animation. . Rolla:: University of Missouri-Rolla;.
  34. Vancíková Z., Dvorák P.. ( 2001;). Cytomegalovirus infection in immunocompetent and immunocompromised individuals – a review. . Curr Drug Targets Immune Endocr Metabol Disord 1:, 179–187. [CrossRef][PubMed]
    [Google Scholar]
  35. Venclovas C., Timinskas A., Siksnys V.. ( 1994;). Five-stranded β-sheet sandwiched with two α-helices: a structural link between restriction endonucleases EcoRI and EcoRV. . Proteins 20:, 279–282. [CrossRef][PubMed]
    [Google Scholar]
  36. Warming S., Costantino N., Court D. L., Jenkins N. A., Copeland N. G.. ( 2005;). Simple and highly efficient BAC recombineering using galK selection. . Nucleic Acids Res 33:, e36. [CrossRef][PubMed]
    [Google Scholar]
  37. Weller S. K., Seghatoleslami M. R., Shao L., Rowse D., Carmichael E. P.. ( 1990;). The herpes simplex virus type 1 alkaline nuclease is not essential for viral DNA synthesis: isolation and characterization of a lacZ insertion mutant. . J Gen Virol 71:, 2941–2952. [CrossRef][PubMed]
    [Google Scholar]
  38. Wing B. A., Huang E. S.. ( 1995;). Analysis and mapping of a family of 3′-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99. . J Virol 69:, 1521–1531.[PubMed]
    [Google Scholar]
  39. Yu D., Silva M. C., Shenk T.. ( 2003;). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. . Proc Natl Acad Sci U S A 100:, 12396–12401. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.034876-0
Loading
/content/journal/jgv/10.1099/vir.0.034876-0
Loading

Data & Media loading...

Supplements

Supplementary material 

PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error