1887

Abstract

The glycoproteins gH and gL of human cytomegalovirus (HCMV) form a complex either with pUL74 (trimeric complex) or with proteins of the UL128 locus (pentameric complex). While the pentameric complex is dispensable for viral growth in fibroblasts, deletion of pUL74 causes a small plaque phenotype in HCMV lab strains, accompanied by greatly reduced cell-free infectivity. As HCMV isolates, shortly after cultivation from clinical specimens, do not release cell-free infectious viruses, we wondered whether deletion of pUL74 would also affect virus growth in this background. To address this question, we took advantage of the bacterial artificial chromosome (BAC)-cloned virus Merlin-RL13tetO, which grows cell associated due to the inducible expression of the viral RL13 gene, thereby resembling clinical isolates. Stop codons were introduced by seamless mutagenesis into UL74 and/or the UL128 locus to prevent expression of the trimeric or pentameric complex, respectively. Virus mutants were reconstituted by transfection of the respective genomes into cultured cells and analysed with respect to focal growth. When the UL128 locus was intact, deletion of pUL74 did not notably affect focal growth of Merlin, irrespective of RL13 expression. In the absence of UL128 expression, foci were increased compared with wild-type, and infectious cell-free virus was produced. Under these conditions, disruption of UL74 completely prevented virus spread from initially transfected cells to surrounding cells. In conclusion the contribution of pUL74 is masked when the UL128 locus is expressed at high levels, and its role in cell-free virus spread is only revealed when expression of the pentameric complex is inhibited.

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2016-08-01
2019-09-23
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References

  1. Adler B. , Scrivano L. , Ruzcics Z. , Rupp B. , Sinzger C. , Koszinowski U. . ( 2006;). Role of human cytomegalovirus UL131A in cell type-specific virus entry and release. . J Gen Virol 87: 2451–2460. [CrossRef] [PubMed]
    [Google Scholar]
  2. Ciferri C. , Chandramouli S. , Donnarumma D. , Nikitin P. A. , Cianfrocco M. A. , Gerrein R. , Feire A. L. , Barnett S. W. , Lilja A. E. et al. ( 2015;). Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes. . Proc Natl Acad Sci USA 112: 1767–1772. [CrossRef] [PubMed]
    [Google Scholar]
  3. Connolly S. A. , Jackson J. O. , Jardetzky T. S. , Longnecker R. . ( 2011;). Fusing structure and function: a structural view of the herpesvirus entry machinery. . Nat Rev Microbiol 9: 369–381. [CrossRef] [PubMed]
    [Google Scholar]
  4. Dargan D. J. , Douglas E. , Cunningham C. , Jamieson F. , Stanton R. J. , Baluchova K. , McSharry B. P. , Tomasec P. , Emery V. C. et al. ( 2010;). Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture. . J Gen Virol 91: 1535–1546. [CrossRef] [PubMed]
    [Google Scholar]
  5. 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 USA 100: 14223–14228. [CrossRef] [PubMed]
    [Google Scholar]
  6. Eisenberg R. J. , Atanasiu D. , Cairns T. M. , Gallagher J. R. , Krummenacher C. , Cohen G. H. . ( 2012;). Herpes virus fusion and entry: a story with many characters. . Viruses 4: 800–832. [CrossRef] [PubMed]
    [Google Scholar]
  7. Fouts A. E. , Comps-Agrar L. , Stengel K. F. , Ellerman D. , Schoeffler A. J. , Warming S. , Eaton D. L. , Feierbach B. . ( 2014;). Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109. . Proc Natl Acad Sci USA 111: 8209–8214. [CrossRef] [PubMed]
    [Google Scholar]
  8. Gatherer D. , Seirafian S. , Cunningham C. , Holton M. , Dargan D. J. , Baluchova K. , Hector R. D. , Galbraith J. , Herzyk P. et al. ( 2011;). High-resolution human cytomegalovirus transcriptome. . Proc Natl Acad Sci USA 108: 19755–19760. [CrossRef] [PubMed]
    [Google Scholar]
  9. Gerna G. , Sarasini A. , Patrone M. , Percivalle E. , Fiorina L. , Campanini G. , Gallina A. , Baldanti F. , Revello M. G. . ( 2008;). Human cytomegalovirus serum neutralizing antibodies block virus infection of endothelial/epithelial cells, but not fibroblasts, early during primary infection. . J Gen Virol 89: 853–865. [CrossRef] [PubMed]
    [Google Scholar]
  10. Hahn G. , Revello M. G. , Patrone M. , Percivalle E. , Campanini G. , Sarasini A. , Wagner M. , Gallina A. , Milanesi G. et al. ( 2004;). Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. . J Virol 78: 10023–10033. [CrossRef] [PubMed]
    [Google Scholar]
  11. Hamprecht K. , Witzel S. , Maschmann J. , Dietz K. , Baumeister A. , Mikeler E. , Goelz R. , Speer C. P. , Jahn G. . ( 2003;). Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation. . J Clin Virol 28: 303–316. [CrossRef] [PubMed]
    [Google Scholar]
  12. Hobom U. , Brune W. , Messerle M. , Hahn G. , Koszinowski U. H. . ( 2000;). Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes. . J Virol 74: 7720–7729.[PubMed] [CrossRef]
    [Google Scholar]
  13. Huber M. T. , Compton T. . ( 1997;). Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex. . J Virol 71: 5391–5398.[PubMed]
    [Google Scholar]
  14. Huber M. T. , Compton T. . ( 1998;). The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex. . J Virol 72: 8191–8197.[PubMed]
    [Google Scholar]
  15. Jäger V. , Büssow K. , Wagner A. , Weber S. , Hust M. , Frenzel A. , Schirrmann T. . ( 2013;). High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. . BMC Biotechnol 13: 52. [CrossRef] [PubMed]
    [Google Scholar]
  16. Jiang X. J. , Adler B. , Sampaio K. L. , Digel M. , Jahn G. , Ettischer N. , Stierhof Y. D. , Scrivano L. , Koszinowski U. et al. ( 2008;). UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions. . J Virol 82: 2802–2812. [CrossRef] [PubMed]
    [Google Scholar]
  17. Lemmermann N. A. , Krmpotic A. , Podlech J. , Brizic I. , Prager A. , Adler H. , Karbach A. , Wu Y. , Jonjic S. et al. ( 2015;). Non-redundant and redundant roles of cytomegalovirus gH/gL complexes in host organ entry and intra-tissue spread. . PLoS Pathog 11: e1004640. [CrossRef] [PubMed]
    [Google Scholar]
  18. Li L. , Nelson J. A. , Britt W. J. . ( 1997;). Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex. . J Virol 71: 3090–3097.[PubMed]
    [Google Scholar]
  19. Mattick C. , Dewin D. , Polley S. , Sevilla-Reyes E. , Pignatelli S. , Rawlinson W. , Wilkinson G. , Dal Monte P. , Gompels U. A. . ( 2004;). Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection. . Virology 318: 582–597. [CrossRef] [PubMed]
    [Google Scholar]
  20. Murrell I. , Tomasec P. , Wilkie G. S. , Dargan D. J. , Davison A. J. , Stanton R. J. . ( 2013;). Impact of sequence variation in the UL128 locus on production of human cytomegalovirus in fibroblast and epithelial cells. . J Virol 87: 10489–10500. [CrossRef] [PubMed]
    [Google Scholar]
  21. Paterson D. A. , Dyer A. P. , Milne R. S. , Sevilla-Reyes E. , Gompels U. A. . ( 2002;). A role for human cytomegalovirus glycoprotein O (gO) in cell fusion and a new hypervariable locus. . Virology 293: 281–294. [CrossRef] [PubMed]
    [Google Scholar]
  22. Rasmussen L. , Geissler A. , Cowan C. , Chase A. , Winters M. . ( 2002;). The genes encoding the gCIII complex of human cytomegalovirus exist in highly diverse combinations in clinical isolates. . J Virol 76: 10841–10848.[PubMed] [CrossRef]
    [Google Scholar]
  23. Ryckman B. J. , Chase M. C. , Johnson D. C. . ( 2008a;). HCMV gH/gL/UL128-131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors. . Proc Natl Acad Sci USA 105: 14118–14123.[CrossRef]
    [Google Scholar]
  24. Ryckman B. J. , Rainish B. L. , Chase M. C. , Borton J. A. , Nelson J. A. , Jarvis M. A. , Johnson D. C. . ( 2008b;). Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells. . J Virol 82: 60–70. [CrossRef] [PubMed]
    [Google Scholar]
  25. Sanchez V. , Greis K. D. , Sztul E. , Britt W. J. . ( 2000;). Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. . J Virol 74: 975–986.[PubMed] [CrossRef]
    [Google Scholar]
  26. Scalzo A. A. , Forbes C. A. , Smith L. M. , Loh L. C. . ( 2009;). Transcriptional analysis of human cytomegalovirus and rat cytomegalovirus homologues of the M73/M73.5 spliced gene family. . Arch Virol 154: 65–75. [CrossRef] [PubMed]
    [Google Scholar]
  27. Scrivano L. , Sinzger C. , Nitschko H. , Koszinowski U. H. , Adler B. . ( 2011;). HCMV spread and cell tropism are determined by distinct virus populations. . PLoS Pathog 7: e1001256. [CrossRef] [PubMed]
    [Google Scholar]
  28. Sinzger C. , Knapp J. , Plachter B. , Schmidt K. , Jahn G. . ( 1997;). Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay. . J Virol Methods 63: 103–112.[PubMed] [CrossRef]
    [Google Scholar]
  29. Sinzger C. , Schmidt K. , Knapp J. , Kahl M. , Beck R. , Waldman J. , Hebart H. , Einsele H. , Jahn G. . ( 1999;). Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. . J Gen Virol 80: 2867–2877. [CrossRef] [PubMed]
    [Google Scholar]
  30. Stagno S. , Reynolds D. W. , Tsiantos A. , Fuccillo D. A. , Long W. , Alford C. A. . ( 1975;). Comparative serial virologic and serologic studies of symptomatic and subclinical congenitally and natally acquired cytomegalovirus infections. . J Infect Dis 132: 568–577.[PubMed] [CrossRef]
    [Google Scholar]
  31. Stanton R. J. , Baluchova K. , Dargan D. J. , Cunningham C. , Sheehy O. , Seirafian S. , McSharry B. P. , Neale M. L. , Davies J. A. et al. ( 2010;). Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. . J Clin Invest 120: 3191–3208. [CrossRef] [PubMed]
    [Google Scholar]
  32. Stanton R. , Westmoreland D. , Fox J. D. , Davison A. J. , Wilkinson G. W. . ( 2005;). Stability of human cytomegalovirus genotypes in persistently infected renal transplant recipients. . J Med Virol 75: 42–46. [CrossRef] [PubMed]
    [Google Scholar]
  33. Tischer B. K. , Smith G. A. , Osterrieder N. . ( 2010;). En passant mutagenesis: a two step markerless red recombination system. . Methods Mol Biol 634: 421–430. [CrossRef] [PubMed]
    [Google Scholar]
  34. Vanarsdall A. L. , Johnson D. C. . ( 2012;). Human cytomegalovirus entry into cells. . Curr Opin Virol 2: 37–42. [CrossRef] [PubMed]
    [Google Scholar]
  35. Vanarsdall A. L. , Chase M. C. , Johnson D. C. . ( 2011;). Human cytomegalovirus glycoprotein gO complexes with gH/gL, promoting interference with viral entry into human fibroblasts but not entry into epithelial cells. . J Virol 85: 11638–11645. [CrossRef] [PubMed]
    [Google Scholar]
  36. Wang D. , Shenk T. . ( 2005;). Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. . Proc Natl Acad Sci USA 102: 18153–18158. [CrossRef] [PubMed]
    [Google Scholar]
  37. Wang D. , Yu Q. C. , Schröer J. , Murphy E. , Shenk T. . ( 2007;). Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells. . Proc Natl Acad Sci USA 104: 20037–20042. [CrossRef] [PubMed]
    [Google Scholar]
  38. Wille P. T. , Knoche A. J. , Nelson J. A. , Jarvis M. A. , Johnson D. C. . ( 2010;). A human cytomegalovirus gO-null mutant fails to incorporate gH/gL into the virion envelope and is unable to enter fibroblasts and epithelial and endothelial cells. . J Virol 84: 2585–2596. [CrossRef] [PubMed]
    [Google Scholar]
  39. Yamane Y. , Furukawa T. , Plotkin S. A. . ( 1983;). Supernatant virus release as a differentiating marker between low passage and vaccine strains of human cytomegalovirus. . Vaccine 1: 23–25. [CrossRef] [PubMed]
    [Google Scholar]
  40. Yu D. , Silva M. C. , Shenk T. . ( 2003;). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. . Proc Natl Acad Sci USA 100: 12396–12401. [CrossRef] [PubMed]
    [Google Scholar]
  41. Zhou M. , Lanchy J. M. , Ryckman B. J. . ( 2015;). Human cytomegalovirus gH/gL/gO promotes the fusion step of entry into all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct mechanism. . J Virol 89: 8999–9009. [CrossRef] [PubMed]
    [Google Scholar]
  42. Zhou M. , Yu Q. , Wechsler A. , Ryckman B. J. . ( 2013;). Comparative analysis of gO isoforms reveals that strains of human cytomegalovirus differ in the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope. . J Virol 87: 9680–9690. [CrossRef] [PubMed]
    [Google Scholar]
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