title: A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection.
abstract: Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency.
authors: Division of Pathway Medicine, Edinburgh Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom.
authors: Kai A
authors: KA
authors: Kropp
authors: Division of Pathway Medicine, Edinburgh Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom.
authors: Wei Yuan
authors: WY
authors: Hsieh
authors: Institut d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain.
authors: Elena
authors: E
authors: Isern
authors: Division of Pathway Medicine, Edinburgh Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom.
authors: Thorsten
authors: T
authors: Forster
authors: Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors: Eva
authors: E
authors: Krause
authors: Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors: Wolfram
authors: W
authors: Brune
authors: Institut d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain.
authors: Ana
authors: A
authors: Angulo
authors: Division of Pathway Medicine, Edinburgh Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom; SynthSys, University of Edinburgh, The King's Buildings, Edinburgh, United Kingdom.
authors: Peter
authors: P
authors: Ghazal
mesh_terms: D000208:Acute Disease; D000818:Animals; D047369:Chromatin Immunoprecipitation; D003587:Cytomegalovirus / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000276:immunology*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral / Q000276:immunology*; D055785:Gene Knockdown Techniques; D015151:Immunoblotting; D007249:Inflammation / Q000276:immunology; D008264:Macrophages / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D051193:Toll-Like Receptors / Q000276:immunology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000276:immunology; D014162:Transfection; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D034741:RNA, Small Interfering; D051193:Toll-Like Receptors; D014764:Viral Proteins