The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription

Abstract

The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses.

Human Cytomegalovirus UL138 Protein Inhibits the STING Pathway and Reduces Interferon Beta mRNA Accumulation during Lytic and Latent Infections

“…The redundancy provided by the multiple cGAS/STING/TBK1 and other IFN-producing pathway inhibitors encoded by HCMV means that inactivating a single STING antagonist amid the background of additional pathway inhibitors results in modest quantitative effects on pathway output. Indeed, of the nine known HCMV STING antagonists, seven have had their effects on IFN-β pathway suppression during infection quantitated by either small interfering RNA (siRNA)-mediated knockdown or with viral mutants, and all seven have shown only small increases in IFN-β mRNA induction in the absence of a single STING antagonist ( 11 – 13 , 18 , 19 , 21 , 22 ), similar in magnitude to effects observed in this study. Thus, our quantitative findings on the effects of UL138 on this pathway align well with previous studies and with the concept of functional redundancy.…”

“…HCMV productively infects highly differentiated cells, such as fibroblasts, macrophages, and epithelial, endothelial, smooth muscle, and dendritic cells ( 8 , 9 ). The cGAS/STING/TBK1 pathway has been shown to mediate an IFN response to HCMV infection in fibroblasts ( 10 – 14 ), endothelial cells ( 15 ), macrophages ( 12 , 16 ), and dendritic cells ( 12 , 16 ). Many viruses encode one or more proteins that inactivate innate immunity pathways ( 17 ), including the cGAS/STING/TBK1 pathway.…”

“…Many viruses encode one or more proteins that inactivate innate immunity pathways ( 17 ), including the cGAS/STING/TBK1 pathway. Indeed, HCMV encodes 9 proteins known to inactivate the cGAS/STING/TBK1 pathway that are among the ∼200 proteins expressed during productive infections of highly differentiated cells: UL31 ( 13 ), UL35 ( 11 ), UL42 ( 12 ), UL48 ( 18 ), UL82 (pp71) ( 19 , 20 ), UL83 (pp65) ( 21 ), UL94 ( 22 ), UL122 (IE2) ( 14 ), and Us9 ( 10 ).…”

Evasion of the Host Immune Response by Betaherpesviruses

“…IRF3-5D, a constitutively active form of IRF, did not exhibit decreased activity in the presence of UL35. Notably, signaling through both cGAS-cGAMP-STING and retinoic acid inducible gene I (RIG-I), an RNA sensing PRR [ 60 ], was inhibited [ 61 ].…”

The Regulatory Network of Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes Pathway in Viral Evasion

“…The HCMV tegument protein UL23 interacts with STAT1 and hinders STAT1 phosphorylation from optimizing subsequent viral dissemination ( Feng et al, 2021 ). UL94 of HCMV represses translocation and dimerization of STING to facilitate virus replication; UL82 and UL35 also antagonize cGAS-STING signaling separately at STING trafficking and TBK1 level ( Fu et al, 2017 ; Fabits et al, 2020 ; Zou et al, 2020 ). Moreover, the HCMV pp65 (pUL83) selectively associates with cGAS and disrupts its following signal transduction with STING, supporting HCMV evading from innate immunity ( Biolatti et al, 2018 ).…”