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• W3092023186 abstract "•Multiplexed proteomic screens reveal regulation of host protein abundance by HSV-1•HSV-1 pUL56 targets host proteins such as GOPC for proteasomal degradation•HSV-1-mediated degradation of GOPC remodels the plasma membrane of infected cells•GOPC is important for cell-surface expression of immune receptor TLR2 in keratinocytes Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells. Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells. Herpesviruses are ubiquitous in the human population and are characterized by an ability to establish lifelong infections. Greater than two-thirds of the world’s population are estimated to be infected with herpes simplex virus 1 (HSV-1) or HSV-2 (Looker et al., 2008Looker K.J. Garnett G.P. Schmid G.P. An estimate of the global prevalence and incidence of herpes simplex virus type 2 infection.Bull. World Health Organ. 2008; 86: 805-812Crossref PubMed Scopus (351) Google Scholar, Looker et al., 2015Looker K.J. Magaret A.S. May M.T. Turner K.M. Vickerman P. Gottlieb S.L. Newman L.M. Global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012.PLoS ONE. 2015; 10: e0140765Crossref PubMed Scopus (278) Google Scholar). These infections are generally asymptomatic or give rise to mild symptoms following viral reactivation (oral or genital sores), although they can cause severe diseases of the eye (herpes keratitis), central nervous system (herpes encephalitis), or systemic infections in those with compromised or immature immune systems (Gnann and Whitley, 2017Gnann Jr., J.W. Whitley R.J. Herpes simplex encephalitis: an update.Curr. Infect. Dis. Rep. 2017; 19: 13Crossref PubMed Scopus (63) Google Scholar; Koujah et al., 2019Koujah L. Suryawanshi R.K. Shukla D. Pathological processes activated by herpes simplex virus-1 (HSV-1) infection in the cornea.Cell. Mol. Life Sci. 2019; 76: 405-419Crossref PubMed Scopus (42) Google Scholar; Pinninti and Kimberlin, 2018Pinninti S.G. Kimberlin D.W. Neonatal herpes simplex virus infections.Semin. Perinatol. 2018; 42: 168-175Crossref PubMed Scopus (38) Google Scholar). The replication cycle of herpesviruses entails a complex and carefully controlled transcriptional cascade of viral genes that function both to generate infectious particles and to modulate host factors. HSV-1 genes are conventionally separated into three broad temporal classes (immediate early, early, and late), where proteins expressed earliest during infection serve as transcription factors and/or modulate the host-cell environment and immune responses, whereas those expressed late are structural components of the virion. The best-studied HSV-1 immunomodulatory proteins are infected-cell protein 0 (ICP0) and virion host shutoff protein (vhs). These proteins are known to change the host-cell proteome by suppressing the expression and/or promoting the degradation of various host proteins (Boutell et al., 2011Boutell C. Cuchet-Lourenço D. Vanni E. Orr A. Glass M. McFarlane S. Everett R.D. A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence.PLoS Pathog. 2011; 7: e1002245Crossref PubMed Scopus (111) Google Scholar; Chelbi-Alix and de Thé, 1999Chelbi-Alix M.K. de Thé H. Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins.Oncogene. 1999; 18: 935-941Crossref PubMed Scopus (257) Google Scholar; Jiang et al., 2016Jiang Z. Su C. Zheng C. Herpes simplex virus 1 tegument protein UL41 counteracts IFIT3 antiviral innate immunity.J. Virol. 2016; 90: 11056-11061Crossref PubMed Scopus (23) Google Scholar; Lees-Miller et al., 1996Lees-Miller S.P. Long M.C. Kilvert M.A. Lam V. Rice S.A. Spencer C.A. Attenuation of DNA-dependent protein kinase activity and its catalytic subunit by the herpes simplex virus type 1 transactivator ICP0.J. Virol. 1996; 70: 7471-7477Crossref PubMed Google Scholar; Lilley et al., 2011Lilley C.E. Chaurushiya M.S. Boutell C. Everett R.D. Weitzman M.D. The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0.PLoS Pathog. 2011; 7: e1002084Crossref PubMed Scopus (91) Google Scholar; Orzalli et al., 2013Orzalli M.H. Conwell S.E. Berrios C. DeCaprio J.A. Knipe D.M. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA.Proc. Natl. Acad. Sci. USA. 2013; 110: E4492-E4501Crossref PubMed Scopus (112) Google Scholar; Su and Zheng, 2017Su C. Zheng C. Herpes simplex virus 1 abrogates the cGAS/STING-mediated cytosolic DNA-sensing pathway via its virion host shutoff protein, UL41.J. Virol. 2017; 91 (e02414-16)Crossref Scopus (75) Google Scholar; Zenner et al., 2013Zenner H.L. Mauricio R. Banting G. Crump C.M. Herpes simplex virus 1 counteracts tetherin restriction via its virion host shutoff activity.J. Virol. 2013; 87: 13115-13123Crossref PubMed Scopus (59) Google Scholar). However, the global temporal effects of HSV-1 replication on the host proteome remain poorly characterized. To date, there has been one large-scale proteomic analysis of HSV-1 infection. This work, performed in fibroblasts, quantified the abundance of approximately 4,000 host proteins and characterized changes in protein post-translational modification following infection (Kulej et al., 2017Kulej K. Avgousti D.C. Sidoli S. Herrmann C. Della Fera A.N. Kim E.T. Garcia B.A. Weitzman M.D. Time-resolved global and chromatin proteomics during herpes simplex virus type 1 (HSV-1) infection.Mol. Cell. Proteomics. 2017; 16: S92-S107Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, the molecular mechanisms underlying these changes were not characterized. Quantitative temporal viromics (QTV) is a method to enable highly multiplexed quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection (Weekes et al., 2014Weekes M.P. Tomasec P. Huttlin E.L. Fielding C.A. Nusinow D. Stanton R.J. Wang E.C.Y. Aicheler R. Murrell I. Wilkinson G.W.G. et al.Quantitative temporal viromics: an approach to investigate host-pathogen interaction.Cell. 2014; 157: 1460-1472Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). QTV employs tandem mass tags (TMTs) and triple-stage mass spectrometry (MS3) to facilitate precise quantitation of each protein, and we have applied this technique to study several viruses including human cytomegalovirus (HCMV), Epstein-Barr virus, vaccinia virus, and BK polyomavirus (Caller et al., 2019Caller L.G. Davies C.T.R. Antrobus R. Lehner P.J. Weekes M.P. Crump C.M. Temporal proteomic analysis of BK polyomavirus infection reveals virus-induced G2 arrest and highly effective evasion of innate immune sensing.J. Virol. 2019; 93 (e00595-19)Crossref PubMed Scopus (7) Google Scholar; Ersing et al., 2017Ersing I. Nobre L. Wang L.W. Soday L. Ma Y. Paulo J.A. Narita Y. Ashbaugh C.W. Jiang C. Grayson N.E. et al.A temporal proteomic map of Epstein-Barr virus lytic replication in B cells.Cell Rep. 2017; 19: 1479-1493Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar; Soday et al., 2019Soday L. Lu Y. Albarnaz J.D. Davies C.T.R. Antrobus R. Smith G.L. Weekes M.P. Quantitative temporal proteomic analysis of vaccinia virus infection reveals regulation of histone deacetylases by an interferon antagonist.Cell Rep. 2019; 27: 1920-1933.e7Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar; Weekes et al., 2014Weekes M.P. Tomasec P. Huttlin E.L. Fielding C.A. Nusinow D. Stanton R.J. Wang E.C.Y. Aicheler R. Murrell I. Wilkinson G.W.G. et al.Quantitative temporal viromics: an approach to investigate host-pathogen interaction.Cell. 2014; 157: 1460-1472Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). We have now performed QTV analysis throughout a single replication cycle of HSV-1 in human keratinocytes, the natural target of HSV-1 lytic infection. At each time point, we quantified almost 7,000 human proteins and >90% of canonical HSV-1 proteins, and we have found evidence for the expression of 17 additional HSV-1 proteins beyond the canonical open reading frames (ORFs). We have identified host proteins that are rapidly degraded by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Further, we demonstrate that GOPC degradation is mediated by the poorly characterized HSV-1 pUL56. Plasma membrane profiling shows that pUL56 reduces the cell-surface abundance of multiple host proteins, including the immune signaling molecule Toll-like receptor 2 (TLR2), and we demonstrate that cell-surface expression of TLR2 requires GOPC. This highlights an unanticipated and highly efficient mechanism whereby HSV-1 specifically targets a cellular trafficking factor in order to manipulate the abundance of host proteins on the surface of infected cells. To construct an unbiased global picture of changes in host and viral proteins throughout the course of HSV-1 infection, we infected an immortalized human keratinocyte cell line (HaCaT) with HSV-1 at a high multiplicity of infection (MOI; 10 plaque-forming units [PFUs]/cell) (Figure 1; Table S1). Immunofluorescence analysis of parallel samples confirmed that >95% of cells were infected (Figure S1A). Ten-plex TMTs and MS3 were used to quantify changes in protein expression over six time points (Figure 1A). A particular advantage of such TMT-based quantitation is the measurement of each protein at every time point. This generated the most complete proteomic dataset examining the lytic replication cycle of HSV-1 to date, quantifying 6,956 human proteins and 67/74 canonical HSV-1 proteins, and provided a global view of changes in protein expression during infection. Temporal analysis of viral protein expression over the whole course of infection can provide a complementary system of protein classification, in addition to enabling direct correlation between viral and cellular protein profiles to give insights into viral-host protein interaction (Soday et al., 2019Soday L. Lu Y. Albarnaz J.D. Davies C.T.R. Antrobus R. Smith G.L. Weekes M.P. Quantitative temporal proteomic analysis of vaccinia virus infection reveals regulation of histone deacetylases by an interferon antagonist.Cell Rep. 2019; 27: 1920-1933.e7Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar; Weekes et al., 2014Weekes M.P. Tomasec P. Huttlin E.L. Fielding C.A. Nusinow D. Stanton R.J. Wang E.C.Y. Aicheler R. Murrell I. Wilkinson G.W.G. et al.Quantitative temporal viromics: an approach to investigate host-pathogen interaction.Cell. 2014; 157: 1460-1472Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). The number of classes of viral protein expression was determined by clustering viral proteins using the K-means method. This identified at least five distinct temporal profiles of viral protein expression (Figures S1B–S1E; Table S1). Furthermore, by searching data against a 6-frame translation of the HSV-1 strain used (KOS), eight putative additional HSV-1 proteins (6FT-ORFs) that increased in abundance over the course of infection were identified in this dataset (Figure S2A; Table S1). HSV-1 infection led to >2-fold downregulation of 496 human proteins and >2-fold upregulation of 34 human proteins. Mock and immediate-early (2 h) infection samples clustered separately from early (4, 6 h) and late (9, 12, 18 h) infection time points. The most extensive changes to the cellular proteome occurred late during infection, as might be expected for a virus with a potent host shutoff activity (Figure 1B). This effect can be observed by a general shift to the left in a scatterplot of fold change (Figure 1C). Multiple host targets known to be specifically downregulated during HSV-1 infection were confirmed, including DNA-PKcs (PRKDC) (Lees-Miller et al., 1996Lees-Miller S.P. Long M.C. Kilvert M.A. Lam V. Rice S.A. Spencer C.A. Attenuation of DNA-dependent protein kinase activity and its catalytic subunit by the herpes simplex virus type 1 transactivator ICP0.J. Virol. 1996; 70: 7471-7477Crossref PubMed Google Scholar; Parkinson et al., 1999Parkinson J. Lees-Miller S.P. Everett R.D. Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase.J. Virol. 1999; 73: 650-657Crossref PubMed Google Scholar), interferon gamma-inducible protein 16 (IFI16) (Orzalli et al., 2012Orzalli M.H. DeLuca N.A. Knipe D.M. Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein.Proc. Natl. Acad. Sci. USA. 2012; 109: E3008-E3017Crossref PubMed Scopus (287) Google Scholar), itchy E3 ubiquitin protein ligase (ITCH) (Ushijima et al., 2010Ushijima Y. Luo C. Kamakura M. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch.Virol. J. 2010; 7: 179Crossref PubMed Scopus (20) Google Scholar), promyelocytic leukemia (PML) (Chelbi-Alix and de Thé, 1999Chelbi-Alix M.K. de Thé H. Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins.Oncogene. 1999; 18: 935-941Crossref PubMed Scopus (257) Google Scholar), tripartite motif-containing 27 (TRIM27) (Conwell et al., 2015Conwell S.E. White A.E. Harper J.W. Knipe D.M. Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.J. Virol. 2015; 89: 220-229Crossref PubMed Scopus (22) Google Scholar), nucleus accumbens-associated 1 (NACC1) (Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar), and MORC family CW-type zinc-finger 3 (MORC3) (Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar) (Figures 1C, 1D, and 2D ; Table S1). Proteomic data were validated by comparison to immunoblot analysis of cells infected for 16 h with three independent strains of HSV-1 and with HSV-2, which suggested that many of the changes observed were conserved phenotypes (Figure 1E). All data are shown in Table S1, in which the “plotter” worksheet facilitates interactive generation of temporal graphs of expression of each of the human or viral proteins quantified. Our data on HSV-1-dependent changes to the cellular proteome were compared to data on HSV-1-dependent changes to the transcriptome (total and newly synthesized RNA) and translatome (ribosome profiling) from a recent study (Rutkowski et al., 2015Rutkowski A.J. Erhard F. L’Hernault A. Bonfert T. Schilhabel M. Crump C. Rosenstiel P. Efstathiou S. Zimmer R. Friedel C.C. Dölken L. Widespread disruption of host transcription termination in HSV-1 infection.Nat. Commun. 2015; 6: 7126Crossref PubMed Scopus (123) Google Scholar) using the latest time points from each dataset to compare the greatest abundance changes (18 h for the proteome and 8 h for the transcriptome/translatome; Table S2). These data confirm a general decrease in both protein and total RNA abundance (Figure 2A). However, the data also suggest the proteins exhibiting the largest decreases in abundance are targeted for specific HSV-1-induced protein degradation, rather than inhibition of transcription or translation (Figure 2A). For example, in HSV-1-infected cells the protein TRIM27 was 22-fold less abundant but TRIM27 total RNA was only 2.2-fold reduced, newly synthesized RNA was just 4.4-fold reduced, and there was a slight increase in ribosome-protected fragments. Table S2 shows the comparison of protein abundance changes at 18 h post infection (hpi) versus total RNA, newly synthesized RNA (4sU), and ribosome profiling data (from Rutkowski et al., 2015Rutkowski A.J. Erhard F. L’Hernault A. Bonfert T. Schilhabel M. Crump C. Rosenstiel P. Efstathiou S. Zimmer R. Friedel C.C. Dölken L. Widespread disruption of host transcription termination in HSV-1 infection.Nat. Commun. 2015; 6: 7126Crossref PubMed Scopus (123) Google Scholar), including a plotter function for host proteins quantified across all four datasets. DAVID software (Huang et al., 2009Huang D.W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (22123) Google Scholar) was used to identify pathways significantly enriched among proteins downregulated >2-fold (Figure 2B). Several of these pathways are known to influence HSV-1 infection, for example cell-cycle-associated proteins such as cyclin-dependent kinases (Schang et al., 1998Schang L.M. Phillips J. Schaffer P.A. Requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription.J. Virol. 1998; 72: 5626-5637Crossref PubMed Google Scholar) and a range of DNA damage response pathways (reviewed in Smith and Weller, 2015Smith S. Weller S.K. HSV-I and the cellular DNA damage response.Future Virol. 2015; 10: 383-397Crossref PubMed Scopus (27) Google Scholar). The ubiquitin-like (Ubl) conjugation pathway was significantly enriched, consistent with the known targeting of certain pathway components by herpesviruses to direct cellular prey for degradation. For example, three SUMO family members were downregulated during infection (the fourth was not quantified) (Figure 2C). Components of each enriched cluster are shown in Table S3. A similar analysis of host proteins upregulated >2-fold did not reveal any enriched clusters. Based on the premise that host proteins downregulated early during viral infection are likely to be enriched in factors with antiviral activity (Nightingale et al., 2018Nightingale K. Lin K.-M. Ravenhill B.J. Davies C. Nobre L. Fielding C.A. Ruckova E. Fletcher-Etherington A. Soday L. Nichols H. et al.High-definition analysis of host protein stability during human cytomegalovirus infection reveals antiviral factors and viral evasion mechanisms.Cell Host Microbe. 2018; 24: 447-460.e11Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we analyzed proteins downregulated >4-fold at the earliest time point after HSV-1 infection (2 hpi; Figures 2D and 2E). Of the six proteins thus identified, four have previously been shown to be reduced significantly in HSV-1-infected cells (methyl-CpG-binding domain protein 1 [MBD1] [Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar], MORC3 [Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar], TRIM27 [Conwell et al., 2015Conwell S.E. White A.E. Harper J.W. Knipe D.M. Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.J. Virol. 2015; 89: 220-229Crossref PubMed Scopus (22) Google Scholar], and zinc-finger protein 462 [ZNF462] [Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar]), of which three were shown to be modulated in an ICP0-dependent manner (MBD1, MORC3, and TRIM27) (Sloan et al., 2015Sloan E. Tatham M.H. Groslambert M. Glass M. Orr A. Hay R.T. Everett R.D. Analysis of the SUMO2 proteome during HSV-1 infection.PLoS Pathog. 2015; 11: e1005059Crossref PubMed Scopus (41) Google Scholar). The other two proteins (senataxin [SETX] and GOPC) have not been previously identified as targets of HSV-1-mediated degradation. ITCH, a member of the NEDD4 family of ubiquitin ligases, was rapidly depleted during HSV-1 infection (Figures 1B–1E). pUL56 proteins from HSV-1 and HSV-2 interact with ITCH and NEDD4, leading to proteasomal degradation of these targets (Ushijima et al., 2008Ushijima Y. Koshizuka T. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus type 2 UL56 interacts with the ubiquitin ligase Nedd4 and increases its ubiquitination.J. Virol. 2008; 82: 5220-5233Crossref PubMed Scopus (34) Google Scholar, Ushijima et al., 2010Ushijima Y. Luo C. Kamakura M. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch.Virol. J. 2010; 7: 179Crossref PubMed Scopus (20) Google Scholar). pUL56 is a tail-anchored type II membrane protein found in purified virions (Koshizuka et al., 2002Koshizuka T. Goshima F. Takakuwa H. Nozawa N. Daikoku T. Koiwai O. Nishiyama Y. Identification and characterization of the UL56 gene product of herpes simplex virus type 2.J. Virol. 2002; 76: 6718-6728Crossref PubMed Scopus (63) Google Scholar) and contains three PPXY motifs that interact with NEDD4, likely by binding to WW domains (Ushijima et al., 2008Ushijima Y. Koshizuka T. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus type 2 UL56 interacts with the ubiquitin ligase Nedd4 and increases its ubiquitination.J. Virol. 2008; 82: 5220-5233Crossref PubMed Scopus (34) Google Scholar). Notably, pUL56 does not contain any lysine residues and is thus likely to be refractory to ubiquitination. To further characterize the cellular binding partners of pUL56, stable isotope labeling of amino acids in cell culture (SILAC) immunoprecipitation-mass spectrometry (IP-MS) analysis was performed using cells expressing GFP-tagged pUL56 or GFP alone (Figures 3A and S3; Table S4). Several members of the NEDD4 family of ubiquitin ligases were enriched in the pUL56 IP, as were multiple trafficking protein particle complex II (TRAPPCII) subunits. Strikingly, GOPC was also identified as a binding partner of pUL56. Co-precipitation assays demonstrated that the purified glutathione S-transferase (GST)-tagged pUL56 cytoplasmic domain (residues 1–207) is capable of binding purified GOPC, confirming that these two proteins interact directly (Figure 3B). The N-terminal coiled-coil domain of GOPC mediates its recruitment to the Golgi via an interaction with golgin-160 (Hicks and Machamer, 2005Hicks S.W. Machamer C.E. Isoform-specific interaction of golgin-160 with the Golgi-associated protein PIST.J. Biol. Chem. 2005; 280: 28944-28951Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), whereas the PDZ domain mediates interactions with C-terminal PDZ-binding motifs of cellular partner proteins (Yao et al., 2001Yao R. Maeda T. Takada S. Noda T. Identification of a PDZ domain containing Golgi protein, GOPC, as an interaction partner of frizzled.Biochem. Biophys. Res. Commun. 2001; 286: 771-778Crossref PubMed Scopus (63) Google Scholar). Truncation of GOPC showed that residues 27–236, comprising the N-terminal coiled-coil region, are sufficient to bind to pUL56 (Figure 3B). IP experiments conducted with cells expressing truncated forms of pUL56 demonstrated that residues 1–157 of pUL56 can mediate efficient binding to GOPC whereas residues 1–104 do not, suggesting that a binding site for GOPC may reside within the 53-amino acid sequence between pUL56 residues 105 and 157 (Figure 3C). Taken together, these results suggest a model whereby pUL56 binds both GOPC and the NEDD4 family of ubiquitin ligases, bringing them in close proximity and thus stimulating the ubiquitination and proteolytic degradation of GOPC. To identify the mechanism of GOPC degradation, cells were infected with wild-type (WT) HSV-1 or HSV-1 lacking expression of pUL56 (ΔUL56). Viruses lacking expression of the viral proteins ICP0 (ΔICP0) or vhs (Δvhs) were also included, as both are known to deplete host proteins. Cells were further treated with or without the proteasomal inhibitor MG132. GOPC was degraded during HSV-1 infection in a pUL56-dependent and MG132-inhibitable fashion, whereas GOPC degradation was independent of both ICP0 and vhs (Figure 4A). Immunofluorescence microscopy further demonstrated pUL56-dependent loss of GOPC in HSV-1-infected cells, which was inhibited by MG132 (Figure 4B). HSV-1 pUL56 contains three PPXY motifs, which mediate interaction with the NEDD4 family of E3 ubiquitin ligases (Ushijima et al., 2010Ushijima Y. Luo C. Kamakura M. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch.Virol. J. 2010; 7: 179Crossref PubMed Scopus (20) Google Scholar). Expression of a GFP-tagged construct by transfection demonstrated that pUL56 is sufficient to cause GOPC degradation in the absence of other HSV-1 factors (Figure 4C). Furthermore, the degradation of GOPC was shown to rely on the PPXY motifs of pUL56, as GOPC was not depleted in cells expressing GFP-tagged pUL56 where all three PPXY motifs have been mutated to AAXA (GFP-pUL56-AAXA; Figure 4C). GFP-pUL56-AAXA simultaneously co-localized with GOPC and TGN46 at a juxtanuclear compartment, suggesting pUL56 and GOPC interact at Golgi membranes, where both proteins are known to localize (Hicks and Machamer, 2005Hicks S.W. Machamer C.E. Isoform-specific interaction of golgin-160 with the Golgi-associated protein PIST.J. Biol. Chem. 2005; 280: 28944-28951Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Koshizuka et al., 2002Koshizuka T. Goshima F. Takakuwa H. Nozawa N. Daikoku T. Koiwai O. Nishiyama Y. Identification and characterization of the UL56 gene product of herpes simplex virus type 2.J. Virol. 2002; 76: 6718-6728Crossref PubMed Scopus (63) Google Scholar). To further test the importance of NEDD4 family E3 ubiquitin ligase binding for GOPC degradation by pUL56, a recombinant virus was generated where all three pUL56 PPXY motifs were mutated to AAXA. This mutant phenocopied the pUL56-deletion virus, failing to degrade GOPC and ITCH (a known pUL56 target; Ushijima et al., 2010Ushijima Y. Luo C. Kamakura M. Goshima F. Kimura H. Nishiyama Y. Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch.Virol. J. 2010; 7: 179Crossref PubMed Scopus (20) Google Scholar), even though pUL56 expression was maintained (Figure 4D). To test our model of pUL56 binding simultaneously to GOPC and NEDD4 family E3 ubiquitin ligases, untagged pUL56 (WT or AAXA) was co-expressed with myc-tagged GOPC plus yellow fluorescent protein (YFP)-tagged WW domains of NEDD4, which interact with PPXY motifs, and cell lysates were subjected to IP analysis with a YFP affinity resin. Capture of the YFP-NEDD4-WW domains efficiently co-precipitated WT pUL56 but not pUL56-AAXA (Figure 4E). Importantly, myc-GOPC was co-precipitated with YFP-NEDD4-WW in the presence of WT pUL56, demonstrating formation of a tripartite complex where binding of GOPC to NEDD4 is mediated by pUL56. Furthermore, IP experiments conducted with cells expressing myc-GOPC and hemagglutinin (HA)-tagged ubiquitin demonstrated a marked increase in ubiquitinated myc-GOPC species precipitated from cells co-expressing WT pUL56 as compared to pUL56-AAXA (Figure 4F). Overall, these data demonstrate that pUL56 recruits NEDD4 family ubiquitin ligases to mediate the ubiquitination and proteasomal degradation of GOPC. The rapid depletion of GOPC from cells during HSV-1 infection implies that removal of this host protein may be important for efficient viral replication. However, growth kinetics of HSV-1 ΔUL56, where endogenous levels of GOPC are ma" @default.
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• W3092023186 creator A5090075258 @default.
• W3092023186 date "2020-10-01" @default.
• W3092023186 modified "2023-10-12" @default.
• W3092023186 title "Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation" @default.
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