Hepatocyte-specific EGFR deletion promotes fibrosis but has no effect on steatosis in fast food diet model of MASLD.

BACKGROUND & AIMS: MASLD has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (i.e. EGFR) deletion in the FFD model. METHODS: EGFRflox/flox mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5-months. RESULTS: Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of TGFß1/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR. CONCLUSIONS: Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene-networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling-pathways.

A Novel Role for the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Delta Isoform in Hepatocellular Proliferation.

The phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform (Pik3cd), usually considered immune-specific, was unexpectedly identified as a gene potentially related to either regeneration and/or differentiation in animals lacking hepatocellular Integrin Linked Kinase (ILK). Since a specific inhibitor (Idelalisib, or CAL101) for the catalytic subunit encoded by Pik3cd (p110δ) has reported hepatotoxicity when used for treating chronic lymphocytic leukemia and other lymphomas, the authors aimed to elucidate whether there is a role for p110δ in normal liver function. To determine the effect on normal liver regeneration, partial hepatectomy (PHx) was performed using mice in which p110δ was first inhibited using CAL101. Inhibition led to over a 50% decrease in proliferating hepatocytes in the first 2 days after PHx. This difference correlated with phosphorylation changes in the HGF and EGF receptors (MET and EGFR, respectively) and NF-κB signaling. Ingenuity Pathway Analyses implicated C/EBPß, HGF, and the EGFR heterodimeric partner, ERBB2, as three of the top 20 regulators downstream of p110δ signaling because their pathways were suppressed in the presence of CAL101 at 1 day post-PHx. A regulatory role for p110δ signaling in mouse and rat hepatocytes through MET and EGFR was further verified using hepatocyte primary cultures, in the presence or absence of CAL101. Combined, these data support a role for p110δ as a downstream regulator of normal hepatocytes when stimulated to proliferate.

Correction: Conditional Genetic Elimination of Hepatocyte Growth Factor in Mice Compromises Liver Regeneration after Partial Hepatectomy.

[This corrects the article DOI: 10.1371/journal.pone.0059836.].

Lymphocyte-Specific Protein-1 Suppresses Xenobiotic-Induced Constitutive Androstane Receptor and Subsequent Yes-Associated Protein-Activated Hepatocyte Proliferation.

Activation of constitutive androstane receptor (CAR) transcription factor by xenobiotics promotes hepatocellular proliferation, promotes hypertrophy without liver injury, and induces drug metabolism genes. Previous work demonstrated that lymphocyte-specific protein-1 (LSP1), an F-actin binding protein and gene involved in human hepatocellular carcinoma, suppresses hepatocellular proliferation after partial hepatectomy. The current study investigated the role of LSP1 in liver enlargement induced by chemical mitogens, a regenerative process independent of tissue loss. 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a direct CAR ligand and strong chemical mitogen, was administered to global Lsp1 knockout and hepatocyte-specific Lsp1 transgenic (TG) mice and measured cell proliferation, hypertrophy, and expression of CAR-dependent drug metabolism genes. TG livers displayed a significant decrease in Ki-67 labeling and liver/body weight ratios compared with wild type on day 2. Surprisingly, this was reversed by day 5, due to hepatocyte hypertrophy. There was no difference in CAR-regulated drug metabolism genes between wild type and TG. TG livers displayed increased Yes-associated protein (YAP) phosphorylation, decreased nuclear YAP, and direct interaction between LSP1 and YAP, suggesting LSP1 suppresses TCPOBOP-driven hepatocellular proliferation, but not hepatocyte volume, through YAP. Conversely, loss of LSP1 led to increased hepatocellular proliferation on days 2, 5, and 7. LSP1 selectively suppresses CAR-induced hepatocellular proliferation, but not drug metabolism, through the interaction of LSP1 with YAP, supporting the role of LSP1 as a selective growth suppressor.

Yes-Associated Protein Is Crucial for Constitutive Androstane Receptor-Driven Hepatocyte Proliferation But Not for Induction of Drug Metabolism Genes in Mice.

BACKGROUND AND AIMS: Constitutive androstane receptor (CAR) agonists, such as 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes without any associated liver injury. Yes-associated protein (Yap) is a key transcription regulator that tightly controls organ size, including that of liver. Our and other previous studies suggested increased nuclear localization and activation of Yap after TCPOBOP treatment in mice and the potential role of Yap in CAR-driven proliferative response. Here, we investigated a direct role of Yap in CAR-driven hepatomegaly and hepatocyte proliferation using hepatocyte-specific Yap-knockout (KO) mice. APPROACH AND RESULTS: Adeno-associated virus 8-thyroxine binding globulin promoter-Cre recombinase vector was injected to Yap-floxed mice for achieving hepatocyte-specific Yap deletion followed by TCPOBOP treatment. Yap deletion did not decrease protein expression of CAR or CAR-driven induction of drug metabolism genes (including cytochrome P450 [Cyp] 2b10, Cyp2c55, and UDP-glucuronosyltransferase 1a1 [Ugt1a1]). However, Yap deletion substantially reduced TCPOBOP-induced hepatocyte proliferation. TCPOBOP-driven cell cycle activation was disrupted in Yap-KO mice because of delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma protein. Furthermore, the induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2, and B1), and important mitotic regulators (such as Aurora B kinase and polo-like kinase 1) was remarkably reduced in Yap-KO mice. Microarray analysis revealed that 26% of TCPOBOP-responsive genes that were mainly related to proliferation, but not to drug metabolism, were altered by Yap deletion. Yap regulated these proliferation genes through alerting expression of Myc and forkhead box protein M1, two critical transcriptional regulators of CAR-mediated hepatocyte proliferation. CONCLUSIONS: Our study revealed an important role of Yap signaling in CAR-driven hepatocyte proliferation; however, CAR-driven induction of drug metabolism genes was independent of Yap.

Phosphorylated Ezrin (Thr567) Regulates Hippo Pathway and Yes-Associated Protein (Yap) in Liver.

The activation of CD81 [the portal of entry of hepatitis C virus (HCV)] by agonistic antibody results in phosphorylation of Ezrin via Syk kinase and is associated with inactivation of the Hippo pathway and increase in yes-associated protein (Yap1). The opposite occurs when glypican-3 or E2 protein of HCV binds to CD81. Hepatocyte-specific glypican-3 transgenic mice have decreased levels of phosphorylated (p)-Ezrin (Thr567) and Yap, increased Hippo activity, and suppressed liver regeneration. The role of Ezrin in these processes has been speculated, but not proved. We show that Ezrin has a direct role in the regulation of Hippo pathway and Yap. Forced expression of plasmids expressing mutant Ezrin (T567D) that mimics p-Ezrin (Thr567) suppressed Hippo activity and activated Yap signaling in hepatocytes in vivo and enhanced activation of pathways of ß-catenin and leucine rich repeat containing G protein-coupled receptor 4 (LGR4) and LGR5 receptors. Hepatoma cell lines JM1 and JM2 have decreased CD81 expression and Hippo activity and up-regulated p-Ezrin (T567). NSC668394, a p-Ezrin (Thr567) antagonist, significantly decreased hepatoma cell proliferation. We additionally show that p-Ezrin (T567) is controlled by epidermal growth factor receptor and MET. Ezrin phosphorylation, mediated by CD81-associated Syk kinase, is directly involved in regulation of Hippo pathway, Yap levels, and growth of normal and neoplastic hepatocytes. The finding has mechanistic and potentially therapeutic applications in hepatocyte growth biology, hepatocellular carcinoma, and HCV pathogenesis.

Correction: Synthesis of IL-6 by Hepatocytes Is a Normal Response to Common Hepatic Stimuli.

[This corrects the article DOI: 10.1371/journal.pone.0096053.].

Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Nonalcoholic Fatty Liver Disease in a Murine Fast-Food Diet Model.

Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoyl-coenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD.

A Noncanonical Role for Plasminogen Activator Inhibitor Type 1 in Obesity-Induced Diabetes.

Obesity is a major risk factor for type 2 diabetes because of chronic hepatic inflammation and resultant insulin resistance. Hepatocyte growth factor (HGF) is responsible for resetting hepatic homeostasis after injury following activation by urokinase-type plasminogen activator (u-PA; encoded by the PLAU gene). Plasminogen activator inhibitor type-1 (PAI-1; encoded by the SERPINE1 gene), a u-PA inhibitor and antifibrinolytic agent, is often elevated in obesity and is linked to cardiovascular events. We hypothesized that, in addition to its role in preventing fibrinolysis, elevated PAI-1 inhibits HGF's activation by u-PA and the resultant anti-inflammatory and hepatoprotective properties. Wild-type and PAI-1 knockout (KO) mice on a high-fat diet both became significantly heavier than lean controls; however, the obese KO mice demonstrated improved glucose metabolism compared with wild-type mice. Obese KO mice also exhibited an increase in conversion of latent single-chain HGF to active two-chain HGF, coinciding with an increase in the phosphorylation of the HGF receptor (HGFR or MET, encoded by the MET gene), as well as dampened inflammation. These results strongly suggest that, in addition to its other functions, PAI-mediated inhibition of HGF activation prohibits the resolution of inflammation in the context of obesity-induced type 2 diabetes.

The histone chaperone HIRA promotes the induction of host innate immune defences in response to HSV-1 infection.

Host innate immune defences play a critical role in restricting the intracellular propagation and pathogenesis of invading viral pathogens. Here we show that the histone H3.3 chaperone HIRA (histone cell cycle regulator) associates with promyelocytic leukaemia nuclear bodies (PML-NBs) to stimulate the induction of innate immune defences against herpes simplex virus 1 (HSV-1) infection. Following the activation of innate immune signalling, HIRA localized at PML-NBs in a Janus-Associated Kinase (JAK), Cyclin Dependent Kinase (CDK), and Sp100-dependent manner. RNA-seq analysis revealed that HIRA promoted the transcriptional upregulation of a broad repertoire of host genes that regulate innate immunity to HSV-1 infection, including those involved in MHC-I antigen presentation, cytokine signalling, and interferon stimulated gene (ISG) expression. ChIP-seq analysis revealed that PML, the principle scaffolding protein of PML-NBs, was required for the enrichment of HIRA onto ISGs, identifying a role for PML in the HIRA-dependent regulation of innate immunity to virus infection. Our data identifies independent roles for HIRA in the intrinsic silencing of viral gene expression and the induction of innate immune defences to restrict the initiation and propagation of HSV-1 infection, respectively. These intracellular host defences are antagonized by the HSV-1 ubiquitin ligase ICP0, which disrupts the stable recruitment of HIRA to infecting viral genomes and PML-NBs at spatiotemporally distinct phases of infection. Our study highlights the importance of histone chaperones to regulate multiple phases of intracellular immunity to virus infection, findings that are likely to be highly pertinent in the cellular restriction of many clinically important viral pathogens.

TCPOBOP-Induced Hepatomegaly and Hepatocyte Proliferation are Attenuated by Combined Disruption of MET and EGFR Signaling.

TCPOBOP (1,4-Bis [2-(3,5-Dichloropyridyloxy)] benzene) is a constitutive androstane receptor (CAR) agonist that induces robust hepatocyte proliferation and hepatomegaly without any liver injury or tissue loss. TCPOBOP-induced direct hyperplasia has been considered to be CAR-dependent with no evidence of involvement of cytokines or growth factor signaling. Receptor tyrosine kinases (RTKs), MET and epidermal growth factor receptor (EGFR), are known to play a critical role in liver regeneration after partial hepatectomy, but their role in TCPOBOP-induced direct hyperplasia, not yet explored, is investigated in the current study. Disruption of the RTK-mediated signaling was achieved using MET knockout (KO) mice along with Canertinib treatment for EGFR inhibition. Combined elimination of MET and EGFR signaling [MET KO + EGFR inhibitor (EGFRi)], but not individual disruption, dramatically reduced TCPOBOP-induced hepatomegaly and hepatocyte proliferation. TCPOBOP-driven CAR activation was not altered in [MET KO + EGFRi] mice, as measured by nuclear CAR translocation and analysis of typical CAR target genes. However, TCPOBOP-induced cell cycle activation was impaired in [MET KO + EGFRi] mice due to defective induction of cyclins, which regulate cell cycle initiation and progression. TCPOBOP-driven induction of FOXM1, a key transcriptional regulator of cell cycle progression during TCPOBOP-mediated hepatocyte proliferation, was greatly attenuated in [MET KO + EGFRi] mice. Interestingly, TCPOBOP treatment caused transient decline in hepatocyte nuclear factor 4 alpha expression concomitant to proliferative response; this was not seen in [MET KO + EGFRi] mice. Transcriptomic profiling revealed the vast majority (~40%) of TCPOBOP-dependent genes primarily related to proliferative response, but not to drug metabolism, were differentially expressed in [MET KO + EGFRi] mice. Conclusion: Taken together, combined disruption of EGFR and MET signaling lead to dramatic impairment of TCPOBOP-induced proliferative response without altering CAR activation.

Lymphocyte-Specific Protein-1 Controls Sorafenib Sensitivity and Hepatocellular Proliferation through Extracellular Signal-Regulated Kinase 1/2 Activation.

The gene leukocyte-specific protein-1 (LSP1), encodes an F-actin binding protein that directly interacts with the mitogen-activated protein kinase pathway. LSP1 has copy number variations in 52% of human hepatocellular carcinoma (HCC). LSP1 suppresses proliferation and migration in hepatocytes. LSP1 binds to the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein/extracellular signal-regulated kinase (ERK)/ERK signaling cassette, the target for sorafenib, a crucial chemotherapeutic agent for HCC. This study addresses the role of LSP1 in liver regeneration and sensitivity to sorafenib in normal and neoplastic hepatocytes. Two mouse models, an Lsp1 global knockout (LSP1KO) and a hepatocyte-specific Lsp1 transgenic (LSP1TG) mouse, were used. After two-thirds hepatectomy (PHx), LSP1KO mice displayed increased proliferation and ERK activation, whereas LSP1TG mice displayed suppressed proliferation and decreased ERK activation. LSP1KO hepatocytes cultured without growth factors exhibited increased proliferation, whereas LSP1TG hepatocytes showed decreased proliferation. Rat and human hepatoma cells expressing Lsp1 shRNA displayed increased sensitivity to sorafenib, as evidenced by decreased cell numbers and phosphorylated ERK expression compared with control. LSP1 KO mice treated with sorafenib before PHx displayed decreased hepatocyte proliferation. Our data show that loss of LSP1 function, observed in HCC, leads to increased sensitivity to sorafenib treatment and enhanced hepatocellular proliferation after PHx in vivo and in cultured cells.

Combined Systemic Disruption of MET and Epidermal Growth Factor Receptor Signaling Causes Liver Failure in Normal Mice.

MET and epidermal growth factor receptor (EGFR) tyrosine kinases are crucial for liver regeneration and normal hepatocyte function. Recently, we demonstrated that in mice, combined inhibition of these two signaling pathways abolished liver regeneration after hepatectomy, with subsequent hepatic failure and death at 15 to 18 days after resection. Morbidity was associated with distinct and specific alterations in important downstream signaling pathways that led to decreased hepatocyte volume, reduced proliferation, and shutdown of many essential hepatocyte functions, such as fatty acid synthesis, urea cycle, and mitochondrial functions. Herein, we explore the role of MET and EGFR signaling in resting mouse livers that are not subjected to hepatectomy. Mice with combined disruption of MET and EGFR signaling were noticeably sick by 10 days and died at 12 to 14 days. Mice with combined disruption of MET and EGFR signaling mice showed decreased liver/body weight ratios, increased apoptosis in nonparenchymal cells, impaired liver metabolic functions, and activation of distinct downstream signaling pathways related to inflammation, cell death, and survival. The present study demonstrates that, in addition to controlling the regenerative response, MET and EGFR synergistically control baseline liver homeostasis in normal mice in such a way that their combined disruption leads to liver failure and death.

Correction: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006769.].

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.

Immunohistochemical Analysis of the Stem Cell Marker LGR5 in Pediatric Liver Disease.

Aims In regenerating liver, hepatic progenitor cells (HPCs) are recruited in response to injury; however, few highly specific human HPC markers exist for the hepatocyte lineage. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a Wnt-associated stem cell marker, has been extensively studied in intestinal stem cells, but little is known about its expression in human liver. We hypothesized that LGR5+ HPCs are induced in the regenerative response to pediatric liver injury. Methods and results Immunohistochemistry was used to characterize LGR5 expression in pediatric liver explants (n = 36). We found cytoplasmic LGR5 expression in all cases; although, much less was observed in acute hepatic necrosis compared to chronic liver diseases. In the latter cases, >50% of hepatocytes were LGR5+, signifying a robust regenerative response mainly in the periphery of regenerative nodules. Only weak LGR5 staining was noted in bile ducts, suggesting hepatocyte-specific expression at the interface. Conclusions Although we observed some degree of regenerative response in all cases, LGR5 was highly expressed in chronic liver disease, possibly due to alternate regeneration and reprogramming pathways. LGR5 is predominant in peri-septal hepatocytes rather than epithelial cell adhesion molecule (EpCAM) positive ductular reactions in chronic pediatric liver diseases and may represent a transitional HPC phenotype for the hepatocyte lineage. These studies are the first to support a unique role for LGR5 in human hepatocyte regeneration and as a potential predictive biomarker for recovery of liver function in children. Future work will also investigate the molecular mechanisms behind LGR5 expression.

Stimulation of the Replication of ICP0-Null Mutant Herpes Simplex Virus 1 and pp71-Deficient Human Cytomegalovirus by Epstein-Barr Virus Tegument Protein BNRF1.

It is now well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx, and ATRX while human cytomegalovirus (HCMV) IE protein IE1 targets PML and Sp100, and its tegument protein pp71 targets hDaxx and ATRX. The functions of these viral regulatory proteins are in part interchangeable; thus, both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by other investigators to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings reinforce the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections. IMPORTANCE: Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen's DNA, while viral functions are expressed that counteract the cell-mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as PML nuclear bodies (PML NBs), or ND10. Members of the alpha-, beta-, and gammaherpesvirus families all express proteins that interact with, degrade, or otherwise counteract the inhibitory effects of various PML NB components. Previous work has shown that there is the potential for a functional interchange between the viral proteins expressed by alpha- and betaherpesviruses, despite a lack of obvious sequence similarity. Here, this concept is extended to include a member of the gammaherpesviruses.

MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus.

UNLABELLED: We previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog 11:e1005059, 2015, http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and γH2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response. IMPORTANCE: Herpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and is a target for HSV-1-mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes, and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100, and γH2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation; therefore, this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation.

Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers. CONCLUSION: MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711-1724).

Analysis of the SUMO2 Proteome during HSV-1 Infection.

Covalent linkage to members of the small ubiquitin-like (SUMO) family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1) ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection.