Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome.

Sara Botto,Daniel N Streblow,Nobuyo Mizuno,Jinu Abraham,Bryan Gall,Klaus J Fruh,Igor Landais,Victor R Defilippis,Kara Pryke

doi:10.1128/mbio.02510-18

Abstract

Secretion of interleukin-1β (IL-1β) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1β following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1β secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1β processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1β gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1β. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1β maturation to prevent this response, we find that HCMV and IE86 target the IL-1β protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1β transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

Highlights

Secretion of interleukin-1␤ (IL-1␤) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form
The mechanistic bases of Human cytomegalovirus (HCMV)-mediated stimulation of signaling pathways that lead to activation of transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor ␬B (NF-␬B), which are involved in the synthesis of antiviral and proinflammatory mRNAs, respectively, have been studied in detail, and crucial roles for double-stranded DNA (dsDNA)-dependent Pattern recognition receptors (PRRs), such as ZBP1/DAI, cGAS, and IFI16, have been reported [35,36,37,38,39,40]
The results presented above demonstrate that live HCMV is capable of inhibiting transcription of pro-IL-1␤ mRNA (Fig. 4C) and that expression of the intermediate early 86-kDa protein (IE86) protein alone associates with strongly diminished secretion of the mature cytokine in response to UV-HCMV exposure (Fig. 5B)

Summary

Introduction

Secretion of interleukin-1␤ (IL-1␤) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Pattern recognition receptors (PRRs) are initially responsible for detecting pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively) and thereby activating innate signaling processes that culminate in the expression of antiviral effectors and secretion of immunologically active factors (reviewed in references 1 and 2). Among these are interleukin-1␤ (IL-1␤) and IL-18, which are synthesized as immature proteins (pro-IL) that are cleaved by the cysteine protease caspase-1. While evidence suggests that AIM2 is involved in HCMV-induced processing of IL-1␤ [41] and that caspase-1 contributes to secretion of the protein from endothelial and smooth muscle cells [33], whether other PRRs or noncanonical inflammasome components are required

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