Human 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.

• Keywords
• host-pathogen interaction, immune evasion, innate immunity, lysosome, proteasome, protein degradation, pulsed SILAC, quantitative proteomics, restriction factor, tandem mass tag,
• MeSH
• Cytomegalovirus Infections genetics   immunology   virology   MeSH
• Cytomegalovirus genetics   immunology   physiology   MeSH
• DNA-Binding Proteins chemistry   genetics   immunology   MeSH
• Immune Evasion MeSH
• Humans MeSH
• Proteins chemistry   genetics   immunology   MeSH
• Proteomics MeSH
• Protein Stability MeSH
• Transcription Factors chemistry   genetics   immunology   MeSH
• Viral Proteins chemistry   genetics   immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• HLTF protein, human MeSH Browser
• Proteins MeSH
• Transcription Factors MeSH
• Viral Proteins MeSH

The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.

• Keywords
• cytomegalovirus, human, immune evasion, immunology, infectious disease, microbiology, natural killer cell, virus,
• MeSH
• Killer Cells, Natural immunology   MeSH
• Cytomegalovirus immunology   pathogenicity   MeSH
• Immune Evasion MeSH
• Immunologic Factors antagonists & inhibitors   MeSH
• Host-Pathogen Interactions * MeSH
• Humans MeSH
• Membrane Proteins antagonists & inhibitors   MeSH
• Proteomics MeSH
• Viral Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Immunologic Factors MeSH
• Membrane Proteins MeSH
• Viral Proteins MeSH