Introduction
Human 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.