The role of Human Cytomegalovirus UL82 regarding the innate immune response

Following cell entry, Human Cytomegalovirus (HCMV) is recognised via the viral Pathogen-Associated Molecular Patterns (PAMPs) by Pattern Recognition Receptors (PRRs), resulting in the activation of a signalling cascade culminating in the expression of type I Interferons (IFNs) and subsequently Interferon Stimulated Genes (ISGs) and induction of an anti-viral state. Consequently, HCMV has evolved means to modulate this early host response to successfully establish an infection. Following infection, the tegument protein UL82 (also known as pp71, encoded by HCMV ORF UL82), translocates to the nucleus and transactivates the HCMV MIEP. UL82 antagonizes DAXX [1], a viral restriction factor in the nucleus and can also localise in the ER antagonising the type I IFN response by interfering with the adaptor protein Stimulator of Interferon Genes (STING) [2] resulting in an inadequate activation of downstream kinase TANK Binding kinase 1 (TBK1) and the transcription factor Interferon Regulatory Factor 3 (IRF3). UL82 has been shown to interact with another tegument protein UL35 and both proteins together enhance the activation of the HCMV Major immediate Early promoter (MIEP) [3]. A previous study demonstrated that UL35 independently antagonises the type I IFN response by interacting with TBK1 [4]. Due to the central role of UL82 and UL35 in initiation of viral gene expression and immune modulation, this study investigated the role of UL82 alone, and in combination with UL35 regarding their effect on the type I IFN response. Firstly, in IFNβ luciferase assays, UL82 alone, and in combination with UL35 inhibited the IFNβ promoter activation at multiple steps: at the level of PRRs, downstream kinase, and transcription factor IRF3 in the nucleus, leaving the NF-κB-mediated promoter activation unaffected. Importantly, UL82 inhibited not only the cGAS-STING-mediated but also RIG-I mediated type I IFN response which is STING adaptor-independent. While UL35 did not individually inhibit downstream of IRF3, UL35, together with UL82 cooperatively inhibited IRF3-mediated IFNβ transcription, changed its subcellular localisation, forming punctate structures in the nucleus while neither of them inhibited IFNAR signalling, as evidenced from ISRE reporter luciferase assays. To further confirm the phenotype in a more physiological context, primary fibroblasts (HFF-1) cells inducibly expressing UL82 and UL35 were used. On activation of PRR signalling, IFNB1 and IFIT3 transcript levels were reduced in the presence of UL82 and upon IFNβ stimulation, IFNAR-dependent ISGs IRF9 and STAT2 transcript levels remained unaffected, congruent with the reporter assays in HEK 293T cells. However, HFF-1 cells inducibly co-expressing UL35+UL82 showed an inconsistent phenotype and their cooperative effect could not be recapitulated in HFF-1 cells. Further, a global RNA sequencing approach with HFF-1 cells expressing UL82 revealed its inhibitory effects on the transcription of antiviral effector proteins and pro-inflammatory cytokines. Upon infection of HFF-1 cells with HCMV wildtype (WT) and UL82stop, IFNB1 transcript levels were elevated in UL82stop-infected HFF-1 cells compared to HCMV WT-infected HFF-1 cells, albeit this difference did not reach statistical significance. In a collaborative effort, structure-guided functional assays based on the UL82 crystal structure with residues identified by our collaborators showed the existence of UL82 as a homodimer when expressed in cells although this capability to dimerize was not important for UL82’s antagonistic activity or interaction with DAXX and UL35. Furthermore, substitution mutations introduced on UL35 and UL82 at their interaction interface led to a strong reduction of their interaction. Delving into the mechanisms of inhibition, DAXX or ATRX were not involved in UL82-mediated inhibition of the type I IFN response. UL82 interacted weakly with the transcription factor IRF3 and mRNA export factor Ribonucleic Acid Export factor (RAE-1). The UL82 crystal structure further enabled identification of its interaction surface with UL35, and also revealed its interaction with RAE-1, a connection that will be analysed in future studies. [1] Hwang and Kalejta 2007 [2] Fu et al. 2017 [3] Schierling et al. 2004 [4] Fabits et al. 2020