IntroductionHuman cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that persistently infects the majority of the human population worldwide (Cannon et al., 2010). Following primary infection under the control of a healthy immune system, a latent infection is established that persists lifelong (Reeves et al., 2005). Although primary infection is mostly asymptomatic in healthy individuals, HCMV may lead to significant morbidity or mortality in immunocompromised patients, particularly transplant recipients and AIDS patients (Griffiths et al., 2015). Vertical transmission of HCMV is a leading cause of congenital infection, resulting in deafness and intellectual disability in newborns (Manicklal et al., 2013). Existing therapies that either target the viral polymerase or terminase are associated with significant toxicity and/or sporadic resistance (El Helou and Razonable, 2019). The identification and characterisation of critical facets of host innate immunity that are targeted for degradation by HCMV proteins thus has important implications for antiviral therapy, since such interactions may be inhibitable by small-molecules, facilitating endogenous inhibition of viral replication (Nathans et al., 2008).HCMV has been reported to disrupt interferon (IFN) production, neutralise the IFN response (Le-Trilling and Trilling, 2015;Goodwin et al., 2018), inhibit natural killer (NK) cell activation (Patel et al., 2018), and avoid T cell surveillance via downregulation of MHC molecules (Jackson et al., 2011). A common final pathway for many host protein targets is proteasomal or lysosomal degradation (Halenius et al., 2015). For example, HCMV facilitates viral replication by degrading components of cellular promyelocytic leukemia nuclear bodies (PML-NB) Sp100, MORC3 and DAXX that act as restriction factors (Kim et al., 2011;Tavalai et al., 2011;Schreiner and Wodrich, 2013;Sloan et al., 2016).We previously developed three orthogonal proteomic/transcriptomic screens to quantify protein degradation early during HCMV infection, identifying 133 degraded proteins that were enriched in antiviral restriction factors. The power of this approach was demonstrated by our identification of helicase-like transcription factor (HLTF) as a novel restriction factor that potently inhibited early viral gene expression and was targeted by the HCMV protein UL145 (Nightingale et al., 2018). However, a global approach to identify the mechanism of HCMV-induced protein degradation is lacking. Our previous study employed the broad, non-selective inhibitor MG132, which is known to affect lysosomal cathepsins in addition to the proteasome (Wiertz et al., 1996), and leupeptin which is a naturally occurring protease inhibitor that can inhibit some proteasomal proteases in addition to the lysosome (Nightingale et al., 2018).In this study, we used the selective proteasome inhibitor bortezomib (Chen et al., 2011) to identify proteins specifically targeted for proteasomal degradation during HCMV infection. This identified that the majority of proteins rescued from degradation by MG132 were also rescued by bortezomib, highlighting the role of viral subversion of the proteasome in immune evasion. Our data additionally provide a shortlist of proteins degraded by the proteasome early during infection that are enriched in known antiviral factors for further investigation.