1887

Abstract

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Funding
This study was supported by the:
  • Rebecca Mason , Royal Free Charity , (Award 180386)
  • John Sinclair , Medical Research Council , (Award MR/S00081X/1)
  • Matthew Reeves , Medical Research Council , (Award MR/RO21384/1)
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/content/journal/jgv/10.1099/jgv.0.001419
2020-05-04
2020-06-04
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References

  1. Dupont L, Reeves MB. Cytomegalovirus latency and reactivation: recent insights into an age old problem. Rev Med Virol 2016; 26:75–89 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  2. Griffiths P, Baraniak I, Reeves M. The pathogenesis of human cytomegalovirus. J Pathol 2015; 235:288–297 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  3. Poole E, Sinclair J. Sleepless latency of human cytomegalovirus. Med Microbiol Immunol 2015; 204:421–429 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  4. Kline JN, Hunninghake GM, He B, Monick MM, Hunninghake GW. Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines. Exp Lung Res 1998; 24:3–14 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  5. Reeves MB, Compton T. Inhibition of inflammatory interleukin-6 activity via extracellular signal-regulated kinase-mitogen-activated protein kinase signaling antagonizes human cytomegalovirus reactivation from dendritic cells. J Virol 2011; 85:12750–12758 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  6. Söderberg-Nauclér C, Fish KN, Nelson JA. Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 1997; 91:119–126 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  7. Hahn G, Jores R, Mocarski ES. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci U S A 1998; 95:3937–3942 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  8. Prösch S, Wuttke R, Krüger DH, Volk H-D. NF-kappaB--a potential therapeutic target for inhibition of human cytomegalovirus (re)activation?. Biol Chem 2002; 383:1601–1609 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  9. Hargett D, Shenk TE. Experimental human cytomegalovirus latency in CD14+ monocytes. Proc Natl Acad Sci U S A 2010; 107:20039–20044 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  10. O'Connor CM, Murphy EA. A myeloid progenitor cell line capable of supporting human cytomegalovirus latency and reactivation, resulting in infectious progeny. J Virol 2012; 86:9854–9865 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  11. Liu R, Baillie J, Sissons JG, Sinclair JH. The transcription factor YY1 binds to negative regulatory elements in the human cytomegalovirus major immediate early enhancer/promoter and mediates repression in non-permissive cells. Nucleic Acids Res 1994; 22:2453–2459 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  12. Reeves MB, MacAry PA, Lehner PJ, Sissons JGP, Sinclair JH. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci U S A 2005; 102:4140–4145 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  13. Krishna BA, Humby MS, Miller WE, O'Connor CM. Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos. Proc Natl Acad Sci U S A 2019; 116:1755–1764 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  14. Keller MJ, Wheeler DG, Cooper E, Meier JL. Role of the human cytomegalovirus major immediate-early promoter's 19-base-pair-repeat cyclic AMP-response element in acutely infected cells. J Virol 2003; 77:6666–6675 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  15. Keller MJ, Wu AW, Andrews JI, McGonagill PW, Tibesar EE et al. Reversal of human cytomegalovirus major immediate-early enhancer/promoter silencing in quiescently infected cells via the cyclic AMP signaling pathway. J Virol 2007; 81:6669–6681 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  16. Kew VG, Yuan J, Meier J, Reeves MB. Mitogen and stress activated kinases act co-operatively with CREB during the induction of human cytomegalovirus immediate-early gene expression from latency. PLoS Pathog 2014; 10:e1004195 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  17. Collins-McMillen D, Rak M, Buehler JC, Igarashi-Hayes S, Kamil JP et al. Alternative promoters drive human cytomegalovirus reactivation from latency. Proc Natl Acad Sci U S A 2019; 116:17492–17497 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  18. Arend KC, Ziehr B, Vincent HA, Moorman NJ. Multiple transcripts encode full-length human cytomegalovirus IE1 and IE2 proteins during lytic infection. J Virol 2016; 90:8855–8865 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  19. Gustems M, Borst E, Benedict CA, Pérez C, Messerle M et al. Regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate NF-kappaB binding sites in the enhancer. J Virol 2006; 80:9899–9904 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  20. Isern E, Gustems M, Messerle M, Borst E, Ghazal P et al. The activator protein 1 binding motifs within the human cytomegalovirus major immediate-early enhancer are functionally redundant and act in a cooperative manner with the NF-{kappa}B sites during acute infection. J Virol 2011; 85:1732–1746 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  21. Dupont L, Du L, Poulter M, Choi S, McIntosh M et al. Src family kinase activity drives cytomegalovirus reactivation by recruiting MOZ histone acetyltransferase activity to the viral promoter. J Biol Chem 2019; 294:12901–12910 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  22. Reeves MB, Sinclair JH. Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo. J Virol 2013; 87:10660–10667 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  23. Poole E, Juss JK, Krishna B, Herre J, Chilvers ER et al. Alveolar Macrophages Isolated Directly From Human Cytomegalovirus (HCMV)-Seropositive individuals are sites of hcmv reactivation in vivo. J Infect Dis 2015; 211:1936–1942 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  24. Reeves MB, Lehner PJ, Sissons JGP, Sinclair JH. An in vitro model for the regulation of human cytomegalovirus latency and reactivation in dendritic cells by chromatin remodelling. J Gen Virol 2005; 86:2949–2954 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  25. Reeves M, Sissons P, Sinclair J. Reactivation of human cytomegalovirus in dendritic cells. Discov Med 2005; 5:170–174[PubMed][PubMed]
    [Google Scholar]
  26. Murphy JC, Fischle W, Verdin E, Sinclair JH. Control of cytomegalovirus lytic gene expression by histone acetylation. Embo J 2002; 21:1112–1120 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  27. Saffert RT, Penkert RR, Kalejta RF. Cellular and viral control over the initial events of human cytomegalovirus experimental latency in CD34+ cells. J Virol 2010; 84:5594–5604 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  28. Krishna BA, Lau B, Jackson SE, Wills MR, Sinclair JH et al. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells. Sci Rep 2016; 6:24674 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  29. Nevels M, Paulus C, Shenk T. Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation. Proc Natl Acad Sci U S A 2004; 101:17234–17239 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  30. Reeves M, Murphy J, Greaves R, Fairley J, Brehm A et al. Autorepression of the human cytomegalovirus major immediate-early promoter/enhancer at late times of infection is mediated by the recruitment of chromatin remodeling enzymes by IE86. J Virol 2006; 80:9998–10009 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  31. Michaelis M, Köhler N, Reinisch A, Eikel D, Gravemann U et al. Increased human cytomegalovirus replication in fibroblasts after treatment with therapeutical plasma concentrations of valproic acid. Biochem Pharmacol 2004; 68:531–538 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  32. Groves IJ, Reeves MB, Sinclair JH. Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic 'pre-immediate-early' repression of viral gene expression mediated by histone post-translational modification. J Gen Virol 2009; 90:2364–2374 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  33. Ioudinkova E, Arcangeletti MC, Rynditch A, De Conto F, Motta F et al. Control of human cytomegalovirus gene expression by differential histone modifications during lytic and latent infection of a monocytic cell line. Gene 2006; 384:120–128 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  34. Rauwel B, Jang SM, Cassano M, Kapopoulou A, Barde I et al. Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch. eLife 2015; 4: [CrossRef][PubMed][PubMed]
    [Google Scholar]
  35. Lee SH, Albright ER, Lee J-H, Jacobs D, Kalejta RF. Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein. Sci Adv 2015; 1:e1501164 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  36. Grey F. Role of microRNAs in herpesvirus latency and persistence. J Gen Virol 2015; 96:739–751 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  37. Poole E, McGregor Dallas SR, Colston J, Joseph RSV, Sinclair J. Virally induced changes in cellular microRNAs maintain latency of human cytomegalovirus in CD34⁺ progenitors. J Gen Virol 2011; 92:1539–1549 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  38. Murphy E, Vanícek J, Robins H, Shenk T, Levine AJ. Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: implications for latency. Proc Natl Acad Sci U S A 2008; 105:5453–5458 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  39. O'Connor CM, Vanicek J, Murphy EA. Host microRNA regulation of human cytomegalovirus immediate early protein translation promotes viral latency. J Virol 2014; 88:5524–5532 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  40. Grey F, Meyers H, White EA, Spector DH, Nelson J. A human cytomegalovirus-encoded microRNA regulates expression of multiple viral genes involved in replication. PLoS Pathog 2007; 3:e163 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  41. O'Connor CM, Nukui M, Gurova KV, Murphy EA. Inhibition of the fact complex reduces transcription from the human cytomegalovirus major immediate early promoter in models of lytic and latent replication. J Virol 2016; 90:4249–4253 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  42. Liu X, Yuan J, Wu AW, McGonagill PW, Galle CS et al. Phorbol ester-induced human cytomegalovirus major immediate-early (Mie) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells. J Virol 2010; 84:8495–8508 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  43. Yuan J, Li M, Torres YR, Galle CS, Meier JL. Differentiation-Coupled induction of human cytomegalovirus replication by Union of the major enhancer retinoic acid, cyclic AMP, and NF-κB response elements. J Virol 2015; 89:12284–12298 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  44. Goodrum FD, Jordan CT, High K, Shenk T. Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency. Proc Natl Acad Sci U S A 2002; 99:16255–16260 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  45. Strobl H, Bello-Fernandez C, Riedl E, Pickl WF, Majdic O et al. Flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions. Blood 1997; 90:1425–1434 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  46. Honess RW, Roizman B. Regulation of herpesvirus macromolecular synthesis. I. cascade regulation of the synthesis of three groups of viral proteins. J Virol 1974; 14:8–19 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  47. Ross L, Guarino LA. Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells. Virology 1997; 232:105–113 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  48. Fenwick ML, Clark J. The effect of cycloheximide on the accumulation and stability of functional alpha-mRNA in cells infected with herpes simplex virus. J Gen Virol 1983; 64:1955–1963 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  49. Preston CM, Rinaldi A, Nicholl MJ. Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis. J Gen Virol 1998; 79 (Pt 1:117–124 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  50. Cherrington JM, Khoury EL, Mocarski ES. Human cytomegalovirus IE2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J Virol 1991; 65:887–896 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  51. Martínez FP, Cruz R, Lu F, Plasschaert R, Deng Z et al. CTCF binding to the first intron of the major immediate early (Mie) gene of human cytomegalovirus (HCMV) negatively regulates MIE gene expression and HCMV replication. J Virol 2014; 88:7389–7401 [CrossRef][PubMed][PubMed]
    [Google Scholar]
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