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.

Cambridge Institute for Medical Research University of Cambridge Hills Road Cambridge CB2 0XY UK

Cardiff University School of Medicine Division of Infection and Immunity Henry Wellcome Building Heath Park Cardiff CF14 4XN UK

Department of Cell Biology Harvard Medical School 240 Longwood Avenue Boston MA 02115 USA

MRC University of Glasgow Centre for Virus Research Sir Michael Stoker Building 464 Bearsden Road Glasgow G61 1QH UK

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty Kopec 7 65653 Brno Czech Republic

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Achar Y.J., Balogh D., Haracska L. Coordinated protein and DNA remodeling by human HLTF on stalled replication fork. Proc. Natl. Acad. Sci. USA. 2011;108:14073–14078. PubMed PMC

Benjamini Y., Hochberg Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B Methodol. 1995;57:289–300.

Blastyak A., Hajdu I., Unk I., Haracska L. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA. Mol. Cell. Biol. 2010;30:684–693. PubMed PMC

Cox J., Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008;26:1367–1372. PubMed

Davison A.J., Holton M., Dolan A., Dargan D.J., Gatherer D., Hayward G.S. Comparative genomics of primate cytomegaloviruses. In: Reddehase M.J., editor. Cytomegaloviruses: from Molecular Pathogenesis to Intervention. Caister Academic Press; 2013. pp. 1–22.

Ding H., Benotmane A.M., Suske G., Collen D., Belayew A. Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene. J. Biol. Chem. 1999;274:19573–19580. PubMed

Dolan A., Cunningham C., Hector R.D., Hassan-Walker A.F., Lee L., Addison C., Dargan D.J., McGeoch D.J., Gatherer D., Emery V.C. Genetic content of wild-type human cytomegalovirus. J. Gen. Virol. 2004;85:1301–1312. PubMed

Duggal N.K., Emerman M. Evolutionary conflicts between viruses and restriction factors shape immunity. Nat. Rev. Immunol. 2012;12:687–695. PubMed PMC

Ersing I., Nobre L., Wang L.W., Soday L., Ma Y., Paulo J.A., Narita Y., Ashbaugh C.W., Jiang C., Grayson N.E. A temporal proteomic map of epstein-barr virus lytic replication in B cells. Cell Rep. 2017;19:1479–1493. PubMed PMC

Fielding C.A., Aicheler R., Stanton R.J., Wang E.C., Han S., Seirafian S., Davies J., McSharry B.P., Weekes M.P., Antrobus P.R. Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation. PLoS Pathog. 2014;10:e1004058. PubMed PMC

Fielding C.A., Weekes M.P., Nobre L.V., Ruckova E., Wilkie G.S., Paulo J.A., Chang C., Suarez N.M., Davies J.A., Antrobus R. Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation. eLife. 2017;6 PubMed PMC

Halenius A., Gerke C., Hengel H. Classical and non-classical MHC I molecule manipulation by human cytomegalovirus: so many targets-but how many arrows in the quiver? Cell. Mol. Immunol. 2015;12:139–153. PubMed PMC

Hrecka K., Hao C., Shun M.C., Kaur S., Swanson S.K., Florens L., Washburn M.P., Skowronski J. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins. Proc. Natl. Acad. Sci. USA. 2016;113:E3921–E3930. PubMed PMC

Hsu J.L., van den Boomen D.J., Tomasec P., Weekes M.P., Antrobus R., Stanton R.J., Ruckova E., Sugrue D., Wilkie G.S., Davison A.J. Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141. PLoS Pathog. 2015;11:e1004811. PubMed PMC

Huang da W., Sherman B.T., Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009;4:44–57. PubMed

Huttlin E.L., Jedrychowski M.P., Elias J.E., Goswami T., Rad R., Beausoleil S.A., Villen J., Haas W., Sowa M.E., Gygi S.P. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell. 2010;143:1174–1189. PubMed PMC

Jackson S., Xiong Y. CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem. Sci. 2009;34:562–570. PubMed PMC

Kim Y.E., Lee J.H., Kim E.T., Shin H.J., Gu S.Y., Seol H.S., Ling P.D., Lee C.H., Ahn J.H. Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression. J. Virol. 2011;85:11928–11937. PubMed PMC

Lahouassa H., Blondot M.L., Chauveau L., Chougui G., Morel M., Leduc M., Guillonneau F., Ramirez B.C., Schwartz O., Margottin-Goguet F. HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages. Proc. Natl. Acad. Sci. USA. 2016;113:5311–5316. PubMed PMC

Li L., He S., Sun J.M., Davie J.R. Gene regulation by Sp1 and Sp3. Biochem. Cell Biol. 2004;82:460–471. PubMed

Matheson N.J., Sumner J., Wals K., Rapiteanu R., Weekes M.P., Vigan R., Weinelt J., Schindler M., Antrobus R., Costa A.S. Cell surface proteomic map of HIV infection reveals antagonism of amino acid metabolism by Vpu and Nef. Cell Host Microbe. 2015;18:409–423. PubMed PMC

McAlister G.C., Nusinow D.P., Jedrychowski M.P., Wuhr M., Huttlin E.L., Erickson B.K., Rad R., Haas W., Gygi S.P. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 2014;86:7150–7158. PubMed PMC

Mocarski E.S., Shenk T., Griffiths P.D., Pass R.F., editors. Cytomegaloviruses. Sixth Edition. Lipincott Williams and Wilkins; 2013.

Nathans R., Cao H., Sharova N., Ali A., Sharkey M., Stranska R., Stevenson M., Rana T.M. Small-molecule inhibition of HIV-1 Vif. Nat. Biotechnol. 2008;26:1187–1192. PubMed PMC

Nichols W.G., Corey L., Gooley T., Davis C., Boeckh M. High risk of death due to bacterial and fungal infection among cytomegalovirus (CMV)-seronegative recipients of stem cell transplants from seropositive donors: evidence for indirect effects of primary CMV infection. J. Infect. Dis. 2002;185:273–282. PubMed

Reeves M.B., MacAry P.A., Lehner P.J., Sissons J.G., Sinclair J.H. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc. Natl. Acad. Sci. USA. 2005;102:4140–4145. PubMed PMC

Rusinova I., Forster S., Yu S., Kannan A., Masse M., Cumming H., Chapman R., Hertzog P.J. Interferome v2.0: an updated database of annotated interferon-regulated genes. Nucleic Acids Res. 2013;41:D1040–D1046. PubMed PMC

Schreiner S., Wodrich H. Virion factors that target Daxx to overcome intrinsic immunity. J. Virol. 2013;87:10412–10422. PubMed PMC

Sheridan P.L., Schorpp M., Voz M.L., Jones K.A. Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter. J. Biol. Chem. 1995;270:4575–4587. PubMed

Sloan E., Orr A., Everett R.D. MORC3, a component of PML nuclear bodies, has a role in restricting herpes simplex virus 1 and human cytomegalovirus. J. Virol. 2016;90:8621–8633. PubMed PMC

Stanton R.J., Baluchova K., Dargan D.J., Cunningham C., Sheehy O., Seirafian S., McSharry B.P., Neale M.L., Davies J.A., Tomasec P. Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. J. Clin. Invest. 2010;120:3191–3208. PubMed PMC

Stanton R.J., McSharry B.P., Rickards C.R., Wang E.C., Tomasec P., Wilkinson G.W. Cytomegalovirus destruction of focal adhesions revealed in a high-throughput Western blot analysis of cellular protein expression. J. Virol. 2007;81:7860–7872. PubMed PMC

Stern-Ginossar N., Weisburd B., Michalski A., Le V.T., Hein M.Y., Huang S.X., Ma M., Shen B., Qian S.B., Hengel H. Decoding human cytomegalovirus. Science. 2012;338:1088–1093. PubMed PMC

Sun Z., Lu Y., Ruan Q., Ji Y., He R., Qi Y., Ma Y., Huang Y. Human cytomegalovirus UL145 gene is highly conserved among clinical strains. J. Biosci. 2007;32:1111–1118. PubMed

Tavalai N., Adler M., Scherer M., Riedl Y., Stamminger T. Evidence for a dual antiviral role of the major nuclear domain 10 component Sp100 during the immediate-early and late phases of the human cytomegalovirus replication cycle. J. Virol. 2011;85:9447–9458. PubMed PMC

Weekes M.P., Tan S.Y.L., Poole E., Talbot S., Antrobus R., Smith D.L., Montag C., Gygi S.P., Sinclair J.H., Lehner P.J. Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection. Science. 2013;340:199–202. PubMed PMC

Weekes M.P., Tomasec P., Huttlin E.L., Fielding C., Nusinow D., Stanton R.J., Wang E.C., Aicheler R., Murrell I., Wilkinson G.W. Quantitative temporal viromics: an approach to investigate host-pathogen interaction. Cell. 2014;157:1460–1472. PubMed PMC

Wiertz E.J., Jones T.R., Sun L., Bogyo M., Geuze H.J., Ploegh H.L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 1996;84:769–779. PubMed

Wisniewski J.R., Hein M.Y., Cox J., Mann M. A “proteomic ruler” for protein copy number and concentration estimation without spike-in standards. Mol. Cell. Proteomics. 2014;13:3497–3506. PubMed PMC

Wu Y., Zhou X., Barnes C.O., DeLucia M., Cohen A.E., Gronenborn A.M., Ahn J., Calero G. The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction. Nat. Struct. Mol. Biol. 2016;23:933–940. PubMed PMC