Advertisement

Archives of Virology

, Volume 161, Issue 5, pp 1273–1284 | Cite as

Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts

  • Feng ZhuEmail author
  • Jian Yuan
  • Hong-Jian Li
  • Zhi-Feng Zeng
  • Zhi-Wen Luo
  • Shi-Qian Li
  • Chi-Qiang He
  • Xue-Fang Jia
  • Xin Zhang
  • Hui Zuo
  • Yi-Min Liu
  • Martin Chang
  • Yue-Qin Li
  • Tian-Hong ZhouEmail author
Original Article

Abstract

Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

Keywords

Bacterial Artificial Chromosome Bacterial Artificial Chromosome Clone HCMV Infection Tegument Protein Laser Scanning Confocal Fluorescence Microscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

Feng Zhu and Jian Yuan contributed equally to this work and should be considered co-first authors. This work was supported by 211 grants of MOE, National Key Basic Research Program of China (2011CB504800), Guangdong Provincial Medical Science Study of China (Grant No. A2014598) and Guangdong Provincial Department of Science and Technology of China (Grant No. 2014A020212028 and Grant No. 2013B021800087).

References

  1. 1.
    Steininger C (2007) Clinical relevance of cytomegalovirus infection in patients with disorders of the immune system. Clin Microbiol Infect 13:953–963CrossRefPubMedGoogle Scholar
  2. 2.
    Gerna G, Baldanti F, Revello MG (2004) Pathogenesis of human cytomegalovirus infection and cellular targets. Hum Immunol 65:381–386CrossRefPubMedGoogle Scholar
  3. 3.
    Chee MS, Bankier AT, Beck S, Bohni R, Brown CM, Cerny R, Horsnell T, Hutchison CA 3rd, Kouzarides T, Martignetti JA et al (1990) Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Topics Microbiol Immunol 154:125–169Google Scholar
  4. 4.
    Nelson JA, Gnann JW Jr, Ghazal P (1990) Regulation and tissue-specific expression of human cytomegalovirus. Curr Topics Microbiol Immunol 154:75–100Google Scholar
  5. 5.
    Cha TA, Tom E, Kemble GW, Duke GM, Mocarski ES, Spaete RR (1996) Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 70:78–83PubMedPubMedCentralGoogle Scholar
  6. 6.
    Novotny J, Rigoutsos I, Coleman D, Shenk T (2001) In silico structural and functional analysis of the human cytomegalovirus (HHV5) genome. J Mol Biol 310:1151–1166CrossRefPubMedGoogle Scholar
  7. 7.
    Chambers J, Angulo A, Amaratunga D, Guo H, Jiang Y, Wan JS, Bittner A, Frueh K, Jackson MR, Peterson PA, Erlander MG, Ghazal P (1999) DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73:5757–5766PubMedPubMedCentralGoogle Scholar
  8. 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 USA 100:14223–14228CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Yu D, Silva MC, Shenk T (2003) Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci USA 100:12396–12401CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Britt WJ, Mach M (1996) Human cytomegalovirus glycoproteins. Intervirology 39:401–412PubMedGoogle Scholar
  11. 11.
    Compton T, Nowlin DM, Cooper NR (1993) Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193:834–841CrossRefPubMedGoogle Scholar
  12. 12.
    Seo JY, Britt WJ (2007) Cytoplasmic envelopment of human cytomegalovirus requires the postlocalization function of tegument protein pp28 within the assembly compartment. J Virol 81:6536–6547CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gibson W (1981) Structural and nonstructural proteins of strain Colburn cytomegalovirus. Virology 111:516–537CrossRefPubMedGoogle Scholar
  14. 14.
    Klupp BG, Mettenleiter TC (1999) Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein. J Virol 73:3014–3022PubMedPubMedCentralGoogle Scholar
  15. 15.
    Landini MP, Ripalti A (1982) A DNA-nicking activity associated with the nucleocapsid of human cytomegalovirus. Arch Virol 73:351–356CrossRefPubMedGoogle Scholar
  16. 16.
    Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG 2nd, Rodland K, Wiley S, Britt W, Shenk T, Smith RD, Nelson JA (2004) Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 78:10960–10966CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Patrone M, Percivalle E, Secchi M, Fiorina L, Pedrali-Noy G, Zoppe M, Baldanti F, Hahn G, Koszinowski UH, Milanesi G, Gallina A (2003) The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection. J Gen Virol 84:3359–3370CrossRefPubMedGoogle Scholar
  18. 18.
    Zhang W, Li H, Li Y, Zeng Z, Li S, Zhang X, Zou Y, Zhou T (2010) Effective inhibition of HCMV UL49 gene expression and viral replication by oligonucleotide external guide sequences and RNase P. Virol J 7:100CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Romanowski MJ, Garrido-Guerrero E, Shenk T (1997) pIRS1 and pTRS1 are present in human cytomegalovirus virions. J Virol 71:5703–5705PubMedPubMedCentralGoogle Scholar
  20. 20.
    Sanchez V, Greis KD, Sztul E, Britt WJ (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 74:975–986CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gibson W (1996) Structure and assembly of the virion. Intervirology 39:389–400PubMedGoogle Scholar
  22. 22.
    Meier JL, Stinski MF (1996) Regulation of human cytomegalovirus immediate-early gene expression. Intervirology 39:331–342PubMedGoogle Scholar
  23. 23.
    Li Y, Altman S (1996) Cleavage by RNase P of gene N mRNA reduces bacteriophage lambda burst size. Nucleic Acids Res 24:835–842CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Chiocca S, Baker A, Cotten M (1997) Identification of a novel antiapoptotic protein, GAM-1, encoded by the CELO adenovirus. J Virol 71:3168–3177PubMedPubMedCentralGoogle Scholar
  25. 25.
    Ma Y, Wang N, Li M, Gao S, Wang L, Ji Y, Qi Y, He R, Sun Z, Ruan Q (2011) An antisense transcript in the human cytomegalovirus UL87 gene region. Virol J 8:515CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Buscher N, Paulus C, Nevels M, Tenzer S, Plachter B (2015) The proteome of human cytomegalovirus virions and dense bodies is conserved across different strains. Med Microbiol Immunol 204:285–293CrossRefPubMedGoogle Scholar
  27. 27.
    Yu X, Shah S, Lee M, Dai W, Lo P, Britt W, Zhu H, Liu F, Zhou ZH (2011) Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus. J Struct Biol 174:451–460CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Kalejta RF (2008) Tegument proteins of human cytomegalovirus. Microbiol Mol Biol Rev 72:249–265 (table of contents) CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Tomtishen JP 3rd (2012) Human cytomegalovirus tegument proteins (pp65, pp71, pp150, pp28). Virol J 9:22CrossRefPubMedGoogle Scholar
  30. 30.
    Arnon TI, Markel G, Mandelboim O (2006) Tumor and viral recognition by natural killer cells receptors. Semin Cancer Biol 16:348–358CrossRefPubMedGoogle Scholar
  31. 31.
    McLaughlin-Taylor E, Pande H, Forman SJ, Tanamachi B, Li CR, Zaia JA, Greenberg PD, Riddell SR (1994) Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol 43:103–110CrossRefPubMedGoogle Scholar
  32. 32.
    Spaete RR, Mocarski ES (1985) Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol 56:135–143PubMedPubMedCentralGoogle Scholar
  33. 33.
    AuCoin DP, Smith GB, Meiering CD, Mocarski ES (2006) Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation. J Virol 80:8199–8210CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Tandon R, Mocarski ES (2008) Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150. J Virol 82:9433–9444CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Feng Zhu
    • 2
    Email author
  • Jian Yuan
    • 3
  • Hong-Jian Li
    • 1
  • Zhi-Feng Zeng
    • 1
  • Zhi-Wen Luo
    • 1
  • Shi-Qian Li
    • 1
  • Chi-Qiang He
    • 1
  • Xue-Fang Jia
    • 1
  • Xin Zhang
    • 1
  • Hui Zuo
    • 2
  • Yi-Min Liu
    • 2
  • Martin Chang
    • 4
  • Yue-Qin Li
    • 1
  • Tian-Hong Zhou
    • 1
    Email author
  1. 1.College of Life Science and TechnologyJinan UniversityGuangzhouChina
  2. 2.Guangzhou Occupational Disease Preventive and Treatment CenterGuangzhou No. 12 Hospital Affiliated to Guangzhou Medical UniversityGuangzhouChina
  3. 3.Department of PathologyGuangdong Key Laboratory for Bioactive Drugs Research Guangdong Pharmaceutical UniversityGuangzhouChina
  4. 4.Center for Musculoskeletal ResearchUniversity of RochesterRochesterUSA

Personalised recommendations