High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms

Abstract

SummaryHuman cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

Human cytomegalovirus protein pUL36: a dual cell death pathway inhibitor

“…MG132 is known to inhibit calpains and lysosomal cathepsins in addition to the proteasome (10), and was used to generate a comprehensive list of proteins targeted for degradation by HCMV rather than to identify proteins specifically degraded by the proteasome. 8,476 human and 186 viral proteins were quantified in two biological replicates (Dataset S1, which includes an interactive 'Plotter').…”

“…From a comparison with our previous study of HCMV-induced protein degradation between 2-24 h (10), seven proteins were degraded with high confidence throughout early and late infection ( Figures 1C, 1D). These proteins included HLTF and Anaphase Promoting Complex subunits 1 and 5 (ANAPC1/5), whose degradation by HCMV has been well characterised (10,19). The effector of necroptosis MLKL was the most significantly downregulated protein at 48 h of HCMV infection and was among proteins most significantly rescued by addition of MG132 ( Figure 1B).…”

“…(E) Examples of the 37 proteins degraded at 48 h that were not confidently degraded at early time points. These comprised: (i) proteins not degraded at early time points (shown in this figure), (ii) proteins that were insufficiently degraded at early time points to pass filtering criteria, and (iii) proteins not quantified in the Nightingale et al study (10).…”

“…We previously took a systematic approach to identifying the viral proteins responsible for the degradation of host factors, employing a panel of recombinant viruses deleted for various blocks of HCMV genes that are non-essential for replication in vitro (10). These viruses included strain AD169, a highly passaged, laboratory-adapted strain that contains a deletion in the UL/b' region (encoding 20 canonical genes, UL133-UL150A), frameshifts in RL5A, RL13 and UL131A, and a non-synonymous substitution in UL36 that inactivates the ability of pUL36 to bind procaspase 8 and inhibit apoptosis (21,22).…”

“…We have shown previously that >900 host proteins are downregulated >3-fold over the course of HCMV infection, with 133 proteins degraded during the early phase, of which 89% are targeted to the proteasome. These data led directly to the identification of candidate natural killer (NK) cell ligands and identified helicase-like transcription factor (HLTF) as a novel antiviral restriction factor (10,11). However, it is not yet known which proteins are degraded later during infection, or throughout a whole infection time-course.…”