Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large, foreign DNA sequences and, as such, are good candidates for both gene delivery and vaccine development. However, many studies have shown that innate immune responses induced by virus vectors can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Furthermore, amplicons are very interesting tools to study innate cellular responses elicited by entry of HSV-1 particles in the absence of any virus gene expression. For these reasons, in this study we characterized the innate antiviral response established in human fibroblasts of limited passage (HFFF-2) infected by amplicons. Our results indicate that infection with amplicons triggered an interferon (IFN)-regulatory factors 3 and 7 (IRF3/7)-dependent antiviral response, rendered the cells resistant to vesicular stomatitis virus infection and induced significant changes in the pattern of cellular gene expression, including the upregulation of Toll-like receptor 3 (TLR3), IRF7 and IFN-stimulated genes (ISGs). In contrast, we observed only a mild and contained type I IFN response in infected cells. Amplicon infection induced nuclear translocation and subsequent degradation of IRF3, without hyperphosphorylation of the protein. Inhibition of endosome-resident TLR signalling by blocking lysosome maturation or the knockdown of TLR3 and 4 did not abolish the cellular response to amplicons, whereas knockdown of IRF3 and 7 inhibited ISG and IFN- expression severely. Therefore, our results confirm the existence of TLR-independent, IRF3/7-dependent activation pathways triggered by HSV-1 particles in human fibroblasts.


Article metrics loading...

Loading full text...

Full text loading...



  1. Chee, A. V. & Roizman, B.(2004). Herpes simplex virus 1 gene products occlude the interferon signaling pathway at multiple sites. J Virol 78, 4185–4196.[CrossRef] [Google Scholar]
  2. Collins, S. E., Noyce, R. S. & Mossman, K. L.(2004). Innate cellular response to virus particle entry requires IRF3 but not virus replication. J Virol 78, 1706–1717.[CrossRef] [Google Scholar]
  3. Compton, T., Kurt-Jones, E. A., Boehme, K. W., Belko, J., Latz, E., Golenbock, D. T. & Finberg, R. W.(2003). Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J Virol 77, 4588–4596.[CrossRef] [Google Scholar]
  4. Cuchet, D., Ferrera, R., Lomonte, P. & Epstein, A. L.(2005). Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICP0 protein. J Gene Med 7, 1187–1199.[CrossRef] [Google Scholar]
  5. Cuchet, D., Potel, C., Thomas, J. & Epstein, A. L.(2007). HSV-1 amplicon vectors: a promising and versatile tool for gene delivery. Expert Opin Biol Ther 7, 975–995.[CrossRef] [Google Scholar]
  6. de Bouteiller, O., Merck, E., Hasan, U. A., Hubac, S., Benguigui, B., Trinchieri, G., Bates, E. E. & Caux, C.(2005). Recognition of double-stranded RNA by human Toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J Biol Chem 280, 38133–38145.[CrossRef] [Google Scholar]
  7. Doyle, S., Vaidya, S., O'Connell, R., Dadgostar, H., Dempsey, P., Wu, T., Rao, G., Sun, R., Haberland, M. & other authors(2002). IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17, 251–263.[CrossRef] [Google Scholar]
  8. Eidson, K. M., Hobbs, W. E., Manning, B. J., Carlson, P. & DeLuca, N. A.(2002). Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection. J Virol 76, 2180–2191.[CrossRef] [Google Scholar]
  9. Everett, R. D., Parada, C., Gripon, P., Sirma, H. & Orr, A.(2008). Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100. J Virol 82, 2661–2672.[CrossRef] [Google Scholar]
  10. Grandvaux, N., Servant, M. J., tenOever, B., Sen, G. C., Balachandran, S., Barber, G. N., Lin, R. & Hiscott, J.(2002). Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes. J Virol 76, 5532–5539.[CrossRef] [Google Scholar]
  11. Guo, J., Peters, K. L. & Sen, G. C.(2000). Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection. Virology 267, 209–219.[CrossRef] [Google Scholar]
  12. He, B., Gross, M. & Roizman, B.(1997). The γ134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94, 843–848.[CrossRef] [Google Scholar]
  13. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H. & other authors(2000). A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745.[CrossRef] [Google Scholar]
  14. Hochrein, H., Schlatter, B., O'Keeffe, M., Wagner, C., Schmitz, F., Schiemann, M., Bauer, S., Suter, M. & Wagner, H.(2004). Herpes simplex virus type-1 induces IFN-α production via Toll-like receptor 9-dependent and -independent pathways. Proc Natl Acad Sci U S A 101, 11416–11421.[CrossRef] [Google Scholar]
  15. Hodge, P. D. & Stow, N. D.(2001). Effects of mutations within the herpes simplex virus type 1 DNA encapsidation signal on packaging efficiency. J Virol 75, 8977–8986.[CrossRef] [Google Scholar]
  16. Imada, K. & Leonard, W. J.(2000). The Jak-STAT pathway. Mol Immunol 37, 1–11.[CrossRef] [Google Scholar]
  17. Johnson, K. E., Song, B. & Knipe, D. M.(2008). Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling. Virology 374, 487–494.[CrossRef] [Google Scholar]
  18. Kadowaki, N., Antonenko, S., Lau, J. Y. & Liu, Y. J.(2000). Natural interferon α/β-producing cells link innate and adaptive immunity. J Exp Med 192, 219–226.[CrossRef] [Google Scholar]
  19. Kashima, T., Vinters, H. V. & Campagnoni, A. T.(1995). Unexpected expression of intermediate filament protein genes in human oligodendroglioma cell lines. J Neuropathol Exp Neurol 54, 23–31.[CrossRef] [Google Scholar]
  20. Kawai, T. & Akira, S.(2006a). Innate immune recognition of viral infection. Nat Immunol 7, 131–137. [Google Scholar]
  21. Kawai, T. & Akira, S.(2006b). TLR signaling. Cell Death Differ 13, 816–825.[CrossRef] [Google Scholar]
  22. Kopp, E. & Medzhitov, R.(2003). Recognition of microbial infection by Toll-like receptors. Curr Opin Immunol 15, 396–401.[CrossRef] [Google Scholar]
  23. Krisky, D. M., Marconi, P. C., Oligino, T. J., Rouse, R. J., Fink, D. J., Cohen, J. B., Watkins, S. C. & Glorioso, J. C. (1998). Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications. Gene Ther 5, 1517–1530.[CrossRef] [Google Scholar]
  24. Krug, A., Luker, G. D., Barchet, W., Leib, D. A., Akira, S. & Colonna, M.(2004). Herpes simplex virus type 1 activates murine natural interferon-producing cells through Toll-like receptor 9. Blood 103, 1433–1437. [Google Scholar]
  25. Kurt-Jones, E. A., Chan, M., Zhou, S., Wang, J., Reed, G., Bronson, R., Arnold, M. M., Knipe, D. M. & Finberg, R. W.(2004). Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis. Proc Natl Acad Sci U S A 101, 1315–1320.[CrossRef] [Google Scholar]
  26. Leonard, W. J.(2001). Role of Jak kinases and STATs in cytokine signal transduction. Int J Hematol 73, 271–277.[CrossRef] [Google Scholar]
  27. Lin, R., Noyce, R. S., Collins, S. E., Everett, R. D. & Mossman, K. L.(2004). The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J Virol 78, 1675–1684.[CrossRef] [Google Scholar]
  28. Lippmann, J., Rothenburg, S., Deigendesch, N., Eitel, J., Meixenberger, K., van Laak, V., Slevogt, H., N'Guessan, P. D., Hippenstiel, S. & other authors(2008). IFNβ responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI). Cell Microbiol 10, 2579–2588.[CrossRef] [Google Scholar]
  29. Lundberg, P., Welander, P., Han, X. & Cantin, E.(2003). Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo. J Virol 77, 11158–11169.[CrossRef] [Google Scholar]
  30. Melchjorsen, J., Siren, J., Julkunen, I., Paludan, S. R. & Matikainen, S.(2006). Induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by ICP27 targeting NF-κB and IRF-3. J Gen Virol 87, 1099–1108.[CrossRef] [Google Scholar]
  31. Melroe, G. T., DeLuca, N. A. & Knipe, D. M.(2004). Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production. J Virol 78, 8411–8420.[CrossRef] [Google Scholar]
  32. Melroe, G. T., Silva, L., Schaffer, P. A. & Knipe, D. M.(2007). Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: potential role in blocking IFN-β induction. Virology 360, 305–321.[CrossRef] [Google Scholar]
  33. Mogensen, T. H. & Paludan, S. R.(2005). Reading the viral signature by Toll-like receptors and other pattern recognition receptors. J Mol Med 83, 180–192.[CrossRef] [Google Scholar]
  34. Mossman, K. L. & Smiley, J. R.(2002). Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication. J Virol 76, 1995–1998.[CrossRef] [Google Scholar]
  35. Mossman, K. L., Macgregor, P. F., Rozmus, J. J., Goryachev, A. B., Edwards, A. M. & Smiley, J. R.(2001). Herpes simplex virus triggers and then disarms a host antiviral response. J Virol 75, 750–758.[CrossRef] [Google Scholar]
  36. Mulvey, M., Camarena, V. & Mohr, I.(2004). Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and γ134.5 gene products. J Virol 78, 10193–10196.[CrossRef] [Google Scholar]
  37. Nicholl, M. J., Robinson, L. H. & Preston, C. M.(2000). Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1. J Gen Virol 81, 2215–2218. [Google Scholar]
  38. Noyce, R. S., Collins, S. E. & Mossman, K. L.(2006). Identification of a novel pathway essential for the immediate-early, interferon-independent antiviral response to enveloped virions. J Virol 80, 226–235.[CrossRef] [Google Scholar]
  39. Olschowka, J. A., Bowers, W. J., Hurley, S. D., Mastrangelo, M. A. & Federoff, H. J.(2003). Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS. Mol Ther 7, 218–227.[CrossRef] [Google Scholar]
  40. Orvedahl, A., Alexander, D., Talloczy, Z., Sun, Q., Wei, Y., Zhang, W., Burns, D., Leib, D. A. & Levine, B.(2007). HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 1, 23–35.[CrossRef] [Google Scholar]
  41. Paladino, P., Cummings, D. T., Noyce, R. S. & Mossman, K. L.(2006). The IFN-independent response to virus particle entry provides a first line of antiviral defense that is independent of TLRs and retinoic acid-inducible gene I. J Immunol 177, 8008–8016.[CrossRef] [Google Scholar]
  42. Porter, I. M. & Stow, N. D.(2004). 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] [Google Scholar]
  43. Preston, C. M., Harman, A. N. & Nicholl, M. J.(2001). Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J Virol 75, 8909–8916.[CrossRef] [Google Scholar]
  44. Randall, R. E. & Goodbourn, S.(2008). Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89, 1–47.[CrossRef] [Google Scholar]
  45. Rasmussen, S. B., Sorensen, L. N., Malmgaard, L., Ank, N., Baines, J. D., Chen, Z. J. & Paludan, S. R.(2007). Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems. J Virol 81, 13315–13324.[CrossRef] [Google Scholar]
  46. Rasmussen, S. B., Jensen, S. B., Nielsen, C., Quartin, E., Kato, H., Chen, Z. J., Silverman, R. H., Akira, S. & Paludan, S. R.(2009). Herpes simplex virus infection is sensed by both Toll-like receptors and retinoic acid-inducible gene-like receptors, which synergize to induce type I interferon production. J Gen Virol 90, 74–78.[CrossRef] [Google Scholar]
  47. Reske, A., Pollara, G., Krummenacher, C., Katz, D. R. & Chain, B. M.(2008). Glycoprotein-dependent and TLR2-independent innate immune recognition of herpes simplex virus-1 by dendritic cells. J Immunol 180, 7525–7536.[CrossRef] [Google Scholar]
  48. Rutz, M., Metzger, J., Gellert, T., Luppa, P., Lipford, G. B., Wagner, H. & Bauer, S.(2004). Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol 34, 2541–2550.[CrossRef] [Google Scholar]
  49. Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X. O. & Chiocca, E. A.(2001). Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome. Mol Ther 3, 591–601.[CrossRef] [Google Scholar]
  50. Samuel, C. E.(2001). Antiviral actions of interferons. Clin Microbiol Rev 14, 778–809.[CrossRef] [Google Scholar]
  51. Schafer, S. L., Lin, R., Moore, P. A., Hiscott, J. & Pitha, P. M.(1998). Regulation of type I interferon gene expression by interferon regulatory factor-3. J Biol Chem 273, 2714–2720.[CrossRef] [Google Scholar]
  52. Sciortino, M. T., Suzuki, M., Taddeo, B. & Roizman, B.(2001). RNAs extracted from herpes simplex virus 1 virions: apparent selectivity of viral but not cellular RNAs packaged in virions. J Virol 75, 8105–8116.[CrossRef] [Google Scholar]
  53. Servant, M. J., Grandvaux, N., tenOever, B. R., Duguay, D., Lin, R. & Hiscott, J.(2003). Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA. J Biol Chem 278, 9441–9447.[CrossRef] [Google Scholar]
  54. Smiley, J. R.(2004). Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase? J Virol 78, 1063–1068.[CrossRef] [Google Scholar]
  55. Sourvinos, G. & Everett, R. D.(2002). Visualization of parental HSV-1 genomes and replication compartments in association with ND10 in live infected cells. EMBO J 21, 4989–4997.[CrossRef] [Google Scholar]
  56. Spaete, R. R. & Frenkel, N.(1982). The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell 30, 295–304.[CrossRef] [Google Scholar]
  57. Stetson, D. B. & Medzhitov, R.(2006). Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24, 93–103.[CrossRef] [Google Scholar]
  58. Suzuki, M., Chiocca, E. A. & Saeki, Y.(2007). Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. Mol Ther 15, 2017–2026.[CrossRef] [Google Scholar]
  59. Takaoka, A., Wang, Z., Choi, M. K., Yanai, H., Negishi, H., Ban, T., Lu, Y., Miyagishi, M., Kodama, T. & other authors(2007). DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448, 501–505.[CrossRef] [Google Scholar]
  60. Wade-Martins, R., Saeki, Y. & Chiocca, E. A.(2003). Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells. Mol Ther 7, 604–612.[CrossRef] [Google Scholar]
  61. Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R. & Paludan, S. R.(2006). Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 80, 5059–5064.[CrossRef] [Google Scholar]
  62. Wietek, C., Miggin, S. M., Jefferies, C. A. & O'Neill, L. A.(2003). Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-kappa. J Biol Chem 278, 50923–50931.[CrossRef] [Google Scholar]
  63. Yang, H., Ma, G., Lin, C. H., Orr, M. & Wathelet, M. G.(2004). Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-β gene promoter. Eur J Biochem 271, 3693–3703.[CrossRef] [Google Scholar]
  64. Yokota, S., Yokosawa, N., Kubota, T., Suzutani, T., Yoshida, I., Miura, S., Jimbow, K. & Fujii, N.(2001). Herpes simplex virus type 1 suppresses the interferon signaling pathway by inhibiting phosphorylation of STATs and Janus kinases during an early infection stage. Virology 286, 119–124.[CrossRef] [Google Scholar]
  65. Zaupa, C., Revol-Guyot, V. & Epstein, A. L.(2003). Improved packaging system for generation of high-level noncytotoxic HSV-1 amplicon vectors using Cre-loxP site-specific recombination to delete the packaging signals of defective helper genomes. Hum Gene Ther 14, 1049–1063.[CrossRef] [Google Scholar]

Data & Media loading...


[ Single PDF file] (67 KB)


[ PDF] (73 KB)


Most cited this month Most Cited RSS feed

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