Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection.

Interferons (IFN) are expressed in and secreted from cells in response to virus infection, and they induce the expression of a variety of genes called interferon-stimulated genes (ISGs) in infected and surrounding cells to block viral infection and limit spread. The mechanisms of action of a number of cytoplasmic ISGs have been well defined, but little is known about the mechanism of action of nuclear ISGs. Constitutive levels of nuclear interferon-inducible protein 16 (IFI16) serve to induce innate signaling and epigenetic silencing of herpes simplex virus (HSV), but only when the HSV infected cell protein 0 (ICP0) E3 ligase, which promotes IFI16 degradation, is inactivated. In this study, we found that following IFN induction, the pool of IFI16 within the infected cell remains high and can restrict wild-type viral gene expression and replication due to both the induced levels of IFI16 and the IFI16-mediated repression of ICP0 levels. Restriction of viral gene expression is achieved by IFI16 promoting the maintenance of heterochromatin on the viral genome, which silences it epigenetically. These results indicate that a nuclear ISG can restrict gene expression and replication of a nuclear DNA virus by maintaining or preventing the removal of repressive heterochromatin associated with the viral genome.

HSV-1 ICP0 dimer domain adopts a novel ß-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

Effects on exocytosis by two HSV-1 mutants unable to block autophagy.

IMPORTANCE: Pathogens often hijack extracellular vesicle (EV) biogenesis pathways for assembly, egress, and cell-to-cell spread. Herpes simplex virus 1 (HSV-1) infection stimulated EV biogenesis through a CD63 tetraspanin biogenesis pathway and these EVs activated antiviral responses in recipient cells restricting the infection. HSV-1 inhibits autophagy to evade the host, and increased CD63 exocytosis could be a coping mechanism, as CD63 is involved in both cargo delivery to lysosomes during autophagy and exocytosis. We analyzed exocytosis after infection with two HSV-1 mutants, a ΔICP34.5 and a ΔICP0, that could not inhibit autophagy. Unlike HSV-1(F), neither of these viruses stimulated increased EV biogenesis through the CD63 pathway. ΔICP34.5 stimulated production of microvesicles and apoptotic bodies that were CD63-negative, while ΔICP0 displayed an overall reduced production of EVs. These EVs activated innate immunity gene expression in recipient cells. Given the potential use of these mutants for therapeutic purposes, the immunomodulatory properties of EVs associated with them may be beneficial.

Neuronal miR-138 Represses HSV-2 Lytic Infection by Regulating Viral and Host Genes with Mechanistic Differences from HSV-1.

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.

Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 0 (ICP0) Targets of Ubiquitination during Productive Infection of Primary Adult Sensory Neurons.

Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons.

Effect of SUMO-SIM Interaction on the ICP0-Mediated Degradation of PML Isoform II and Its Associated Proteins in Herpes Simplex Virus 1 Infection.

ND10 nuclear bodies, as part of the intrinsic defenses, impose repression on incoming DNA. Infected cell protein 0 (ICP0), an E3 ubiquitin ligase of herpes simplex virus 1 (HSV-1), can derepress viral genes by degrading ND10 organizers to disrupt ND10. These events are part of the initial tug of war between HSV-1 and host, which determines the ultimate outcome of infection. Previously, we reported that ICP0 differentially recognizes promyelocytic leukemia (PML) isoforms. ICP0 depends on a SUMO-interaction motif located at residues 362 to 364 (SIM362-364) to trigger the degradation of PML isoforms II, IV, and VI, while using a bipartite sequence flanking the RING domain to degrade PML I. In this study, we investigated how the SUMO-SIM interaction regulates the degradation of PML II and PML II-associated proteins in ND10. We found that (i) the same regulatory mechanism for PML II degradation was detected in cells permissive or nonpermissive to the ICP0-null virus; (ii) the loss of a single SIM362-364 motif was restored by the presence of four consecutive SIMs from RNF4, but was not rescued by only two of the RNF4 SIMs; (iii) the loss of three C-terminal SIMs of ICP0 was fully restored by four RNF4 SIMs and also partially rescued by two RNF4 SIMs; and (iv) a PML II mutant lacking both lysine SUMOylation and SIM was not recognized by ICP0 for degradation, but was localized to ND10 and mitigated the degradation of other ND10 components, leading to delayed viral production. Taken together, SUMO regulates ICP0 substrate recognition via multiple fine-tuned mechanisms in HSV-1 infection.IMPORTANCE HSV-1 ICP0 is a multifunctional immediate early protein key to effective replication in the HSV-1 lytic cycle and reactivation in the latent cycle. ICP0 transactivates gene expression by orchestrating an overall mitigation in host intrinsic/innate restrictions. How ICP0 coordinates its multiple active domains and its diverse protein-protein interactions is a key question in understanding the HSV-1 life cycle and pathogenesis. The present study focuses on delineating the regulatory effects of the SUMO-SIM interaction on ICP0 E3 ubiquitin ligase activity regarding PML II degradation. For the first time, we discovered the importance of multivalency in the PML II-ICP0 interaction network and report the involvement of different regulatory mechanisms in PML II recognition by ICP0 in HSV-1 infection.

The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection.

Herpes simplex virus 1 (HSV-1) infects mucosal epithelial cells and establishes lifelong infections in sensory neurons. Following reactivation, the virus is transferred anterograde to the initial site of infection or to sites innervated by infected neurons, causing vesicular lesions. Upon immunosuppression, frequent HSV-1 reactivation can cause severe diseases, such as blindness and encephalitis. Autophagy is a process whereby cell components are recycled, but it also serves as a defense mechanism against pathogens. HSV-1 is known to combat autophagy through the functions of the γ134.5 protein, which prevents formation of the autophagophore by binding to Beclin 1, a key factor involved in the elongation of the isolation membrane, and by redirecting the protein phosphatase 1α (PP1α) to dephosphorylate the translation initiation factor 2α (eIF2α) to prevent host translational shutoff. Other viral proteins that counteract innate immunity negatively impact autophagy. Here, we present a novel strategy of HSV-1 to evade the host through the downregulation of the autophagy adaptor protein sequestosome (p62/SQSTM1) and of the mitophagy adaptor optineurin (OPTN). This down-modulation occurs during the early steps of the infection. We also found that infected cell protein 0 (ICP0) of the virus mediates the down-modulation of the two autophagy adaptors in a mechanism independent of its E3 ubiquitin ligase activity. Cells depleted of either p62 or OPTN were able to mount greater antiviral responses, whereas cells expressing exogenous p62 displayed decreased virus yields. We conclude that downregulation of p62/SQSTM1 and OPTN is a viral strategy to counteract the host.IMPORTANCE Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves downregulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question of why the virus targets two major autophagy adaptors if it has mechanisms to block autophagosome formation. P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting, and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 may have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus may target another antiviral function of these proteins.

Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay.

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-ß)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments.

Nuclear domain 10 (ND10) bodies are small (0.1-1 µM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-ß. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-ß in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-ß and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.

HSV-1 triggers paracrine fibroblast growth factor response from cortical brain cells via immediate-early protein ICP0.

BACKGROUND: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells. METHOD: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells. RESULTS: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism. CONCLUSIONS: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0.IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in HSV-1 infection, we investigated the potential players involved in this nuclear-to-cytoplasmic translocation. We found that there is a nuclear retention force in an ICP0 E3 ubiquitin ligase-dependent manner. In addition, we identified the C terminus of ICP0 as a cis element cooperating with late viral proteins to overcome the nuclear retention and stimulate the nuclear-to-cytoplasmic translocation of ICP0.

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression.

Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.

Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0.

To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.

PML plays both inimical and beneficial roles in HSV-1 replication.

After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1-1 µM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-ß. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-ß is significantly more effective in blocking viral replication in murine PML(+/+) cells than in sibling PML(-/-) cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-ß. To define more precisely the function of PML in HSV-1 replication, we constructed a PML(-/-) human cell line. We report that in PML(-/-) cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML(-/-) cells infected at 5 pfu wild-type virus per cell, but poorly in PML(-/-) cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML(-/-) infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-ß effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus.

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway.IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

Cbl E3 Ligase Mediates the Removal of Nectin-1 from the Surface of Herpes Simplex Virus 1-Infected Cells.

The Cbl E3 ligase has been linked to the down-modulation of surface signaling responses by inducing internalization of surface receptors. The adaptor protein CIN85 is a partner of Cbl that augments many of these interactions. Previously, an interaction was demonstrated between ICP0 and CIN85, which results in the removal of epidermal growth factor receptor (EGFR) from the surface of the infected cells with a concomitant attenuation of EGFR signaling. Here, we examined whether Cbl mediates the removal of the herpes simplex virus 1 (HSV-1) entry receptor Nectin-1 from the surface of infected cells. We found the following: (i) that Cbl, Nectin-1, and the viral glycoprotein D (gD) form a complex in infected cells; (ii) that during infection Nectin-1 is removed from the surface of the infected cells but is retained on the surface of cells that have been depleted of Cbl; and (iii) that in cells infected with a ΔICP0 mutant virus, Nectin-1 remained on the cell surface. Thus, Cbl is necessary but not sufficient for the removal of Nectin-1 from the cell surface. In addition, we observed that in Cbl-depleted cells there was enhanced entry after infection. These cells were susceptible to secondary infections by HSV-1. Viral entry in CIN85-depleted cells was only moderately enhanced compared to that in the Cbl-depleted cells, suggesting that the Cbl-Nectin-1 interaction is likely the key to the downregulation of surface Nectin-1. The removal of the HSV-1 entry receptor Nectin-1 from the surface of the infected cells may be part of the strategy of the virus to efficiently spread to uninfected cells.IMPORTANCE The Cbl E3 ligase suppresses surface signaling responses by inducing internalization of surface components. The targets of Cbl include such components as immune system receptors, growth factor receptors, adhesion, and cell-to-cell contact molecules. The immediate early protein ICP0 of herpes simplex virus 1 (HSV-1) interacts with CIN85, an adaptor protein that augments Cbl functions. The consequence of this interaction is the removal of the epidermal growth factor receptor (EGFR) from the surface of the infected cells with concomitant suppression of the EGF ligand signaling. The viral entry receptor Nectin-1 is also internalized during HSV-1 infection in a Cbl-dependent mechanism, and that increases the opportunity of the virus to spread to uninfected cells. The diversion of the Cbl/CIN85 endocytic machinery may be a strategy utilized by the virus to alter the cell surface pattern to prevent detrimental host responses.

Role of Herpes Simplex Virus 1 γ34.5 in the Regulation of IRF3 Signaling.

During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are among the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (ΔXX) that lacks amino acids 87 to 106, a portion of the previously described TBK1-binding domain of the γ34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284:1097-1105, 2009, https://doi.org/10.1074/JBC.M805905200). These 20 residues are outside the γ34.5 beclin1-binding domain (BBD) that interacts with beclin1 and regulates autophagy. Unexpectedly, ΔXX showed no deficit in replication in vivo in a variety of tissues and showed virulence comparable to that of wild-type and marker-rescued viruses following intracerebral infection. ΔXX was fully capable of mediating the dephosphorylation of eIF2α, and the virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in γ34.5 failed to control IRF3 phosphorylation due to an inability of the mutant to sustain expression of ICP0. Our data show that while γ34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation or on replication and pathogenesis in mice.IMPORTANCE Interferons (IFNs) are potent activators of a variety of host responses that serve to control virus infections. The Herpesviridae have evolved countermeasures to IFN responses. Herpes simplex virus 1 (HSV-1) encodes the multifunctional neurovirulence protein ICP34.5. In this study, we investigated the biological relevance of the interaction between ICP34.5 and TANK-binding kinase 1 (TBK1), an activator of IFN responses. Here, we establish that although ICP34.5 binds TBK1 under certain conditions through a TBK1-binding domain (TBD), there was no direct impact of the TBD on viral replication or virulence in mice. Furthermore, we showed that activation of IRF3, a substrate of TBK1, was independent of the TBD. Instead, we provided evidence that the ability of ICP34.5 to control IRF3 activation is through its ability to reverse translational shutoff and sustain the expression of other IFN inhibitors encoded by the virus. This work provides new insights into the immunomodulatory functions of ICP34.5.

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

Role of ND10 nuclear bodies in the chromatin repression of HSV-1.

Herpes simplex virus (HSV) is a neurotropic virus that establishes lifelong latent infection in human ganglion sensory neurons. This unique life cycle necessitates an intimate relation between the host defenses and virus counteractions over the long course of infection. Two important aspects of host anti-viral defense, nuclear substructure restriction and epigenetic chromatin regulation, have been intensively studied in the recent years. Upon viral DNA entering the nucleus, components of discrete nuclear bodies termed nuclear domain 10 (ND10), converge at viral DNA and place restrictions on viral gene expression. Meanwhile the infected cell mobilizes its histones and histone-associated repressors to force the viral DNA into nucleosome-like structures and also represses viral transcription. Both anti-viral strategies are negated by various HSV countermeasures. One HSV gene transactivator, infected cell protein 0 (ICP0), is a key player in antagonizing both the ND10 restriction and chromatin repression. On one hand, ICP0 uses its E3 ubiquitin ligase activity to target major ND10 components for proteasome-dependent degradation and thereafter disrupts the ND10 nuclear bodies. On the other hand, ICP0 participates in de-repressing the HSV chromatin by changing histone composition or modification and therefore activates viral transcription. Involvement of a single viral protein in two seemingly different pathways suggests that there is coordination in host anti-viral defense mechanisms and also cooperation in viral counteraction strategies. In this review, we summarize recent advances in understanding the role of chromatin regulation and ND10 dynamics in both lytic and latent HSV infection. We focus on the new observations showing that ND10 nuclear bodies play a critical role in cellular chromatin regulation. We intend to find the connections between the two major anti-viral defense pathways, chromatin remodeling and ND10 structure, in order to achieve a better understanding of how host orchestrates a concerted defense and how HSV adapts with and overcomes the host immunity.

A quantitative assay to monitor HSV-1 ICP0 ubiquitin ligase activity in vitro.

The ubiquitin-proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.