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

2020 ◽  
Vol 94 (12) ◽  
Author(s):  
Behdokht Jan Fada ◽  
Elie Kaadi ◽  
Subodh Kumar Samrat ◽  
Yi Zheng ◽  
Haidong Gu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Nuclear Bodies ◽  
Lytic Cycle ◽  
Herpes Simplex Virus 1 ◽  
Associated Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT 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.

scholarly journals A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1

2016 ◽  
Vol 90 (23) ◽  
pp. 10875-10885 ◽  
Author(s):  
Yi Zheng ◽  
Subodh Kumar Samrat ◽  
Haidong Gu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cell Protein ◽  
Physical Interaction ◽  
Herpes Simplex Virus 1 ◽  
C Terminus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTInfected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an α gene product required for viral replication at low multiplicities of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase that degrades the ND10 organizer PML and disperses ND10 to alleviate the repression. In the present study, we focused on understanding the regulation of ICP0 E3 ligase activity in the degradation of different ICP0 substrates. We report the following. (i) A SUMO interaction motif located at ICP0 residues 362 to 364 is required for the degradation of PML isoforms II, IV, and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell's permissiveness to the ICP0-null virus. (ii) Physical interaction between SIM362–364and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM362–364and distal sequences located at the ICP0 C terminus enhance the degradation of PML II. (iii) The ICP0 C terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10 fusion ability, affects the rate of PML I degradation. Taken together, our results show that ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to the different roles these substrates may play in HSV-1 infection.IMPORTANCEViruses have a limited genetic coding capacity but must encounter a multilayered comprehensive host defense. To establish a successful infection, viruses usually produce multifunctional proteins to coordinate the counteractions. Here we report that an HSV-1 protein, ICP0, can recognize individual host factors and target them differently for destruction. We identified elements that are important for the ICP0 E3 ubiquitin ligase to differentially recognize two of its substrates, PML I and PML II. This is the first study that has systematically investigated how ICP0 discriminates two similar molecules by very different mechanisms. This work lays the foundation for understanding the role of host defensive factors and the mechanisms viruses use to take advantage of some host proteins while destroying others.

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

2016 ◽  
Vol 90 (19) ◽  
pp. 8621-8633 ◽  
Author(s):  
Elizabeth Sloan ◽  
Anne Orr ◽  
Roger D. Everett
Keyword(s):  
Immune Response ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Hcmv Infection ◽  
Nuclear Bodies ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Antiviral Role ◽  
Hsv 1

ABSTRACTWe 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 Pathog11: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.IMPORTANCEHerpesviruses 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.

scholarly journals Identification of Three Redundant Segments Responsible for Herpes Simplex Virus 1 ICP0 To Fuse with ND10 Nuclear Bodies

2015 ◽  
Vol 89 (8) ◽  
pp. 4214-4226 ◽  
Author(s):  
Yi Zheng ◽  
Haidong Gu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infected Cell ◽  
Nuclear Bodies ◽  
Cell Protein ◽  
Herpes Simplex Virus 1 ◽  
Infected Cell Protein ◽  
Simplex Virus ◽  
Hsv 1 ◽  
Infected Cell Protein 0

ABSTRACTInfected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a key regulator in both lytic and latent infections. In lytic infection, an important early event is the colocalization of ICP0 to nuclear domain 10 (ND10), the discrete nuclear bodies that impose restrictions on viral expression. ICP0 contains an E3 ubiquitin ligase that degrades promyelocytic leukemia protein (PML) and Sp100, two major components of ND10, and disperses ND10 to alleviate repression. We previously reported that the association between ICP0 and ND10 is a dynamic process that includes three steps: adhesion, fusion, and retention. ICP0 residues 245 to 474, defined as ND10 entry signal (ND10-ES), is a region required for the fusion step. Without ND10-ES, ICP0 adheres at the ND10 surface but fails to enter. In the present study, we focus on characterizing ND10-ES. Here we report the following. (i) Fusion of ICP0 with ND10 relies on specific sequences located within ND10-ES. Replacement of ND10-ES by the corresponding region from ORF61 of varicella-zoster virus did not rescue ND10 fusion. (ii) Three tandem ND10 fusion segments (ND10-FS1, ND10-FS2, and ND10-FS3), encompassing 200 amino acids within ND10-ES, redundantly facilitate fusion. Each of the three segments is sufficient to independently drive the fusion process, but none of the segments by themselves are necessary for ND10 fusion. Only when all three segments are deleted is fusion blocked. (iii) The SUMO interaction motif located within ND10-FS2 is not required for ND10 fusion but is required for the complete degradation of PML, suggesting that PML degradation and ND10 fusion are regulated by different molecular mechanisms.IMPORTANCEND10 nuclear bodies are part of the cell-intrinsic antiviral defenses that restrict viral gene expression upon virus infection. As a countermeasure, infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) localizes to ND10s, degrades the ND10 organizer, and disperses ND10 components in order to alleviate repression. We studied the ICP0-ND10 association to delineate elements important for this dynamic interaction and to understand its role in viral replication and host defense. In this work, we show that ICP0 contains three redundant segments to ensure an effective mergence of ICP0 with ND10 nuclear bodies. This is the first study to systematically investigate ICP0 elements that are important for ICP0-ND10 fusion.

scholarly journals Analysis of the Functional Interchange between the IE1 and pp71 Proteins of Human Cytomegalovirus and ICP0 of Herpes Simplex Virus 1

2014 ◽  
Vol 89 (6) ◽  
pp. 3062-3075 ◽  
Author(s):  
Yongxu Lu ◽  
Roger D. Everett
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Human Cytomegalovirus ◽  
Nuclear Bodies ◽  
Herpes Simplex Virus 1 ◽  
Pml Nuclear Bodies ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHuman cytomegalovirus (HCMV) immediate early protein IE1 and the tegument protein pp71 are required for efficient infection. These proteins have some functional similarities with herpes simplex virus 1 (HSV-1) immediate early protein ICP0, which stimulates lytic HSV-1 infection and derepresses quiescent HSV-1 genomes. All three proteins counteract antiviral restriction mediated by one or more components of promyelocytic leukemia (PML) nuclear bodies, and IE1 and pp71, acting together, almost completely complement ICP0 null mutant HSV-1. Here, we investigated whether ICP0 might substitute for IE1 or pp71 during HCMV infection. Using human fibroblasts that express ICP0, IE1, or pp71 in an inducible manner, we found that ICP0 stimulated replication of both wild-type (wt) and pp71 mutant HCMV while IE1 increased wt HCMV plaque formation and completely complemented the IE1 mutant. Although ICP0 stimulated IE2 expression from IE1 mutant HCMV and increased the number of IE2-positive cells, it could not compensate for IE1 in full lytic replication. These results are consistent with previous evidence that both IE1 and IE2 are required for efficient HCMV gene expression, but they also imply that IE2 functionality is influenced specifically by IE1, either directly or indirectly, and that IE1 may include sequences that have HCMV-specific functions. We discovered a mutant form of IE1 (YL2) that fails to stimulate HCMV infection while retaining 30 to 80% of the activity of the wt protein in complementing ICP0 null mutant HSV-1. It is intriguing that the YL2 mutation is situated in the region of IE1 that is shared with IE2 and which is highly conserved among primate cytomegaloviruses.IMPORTANCEHerpesvirus gene expression can be repressed by cellular restriction factors, one group of which is associated with structures known as ND10 or PML nuclear bodies (PML NBs). Regulatory proteins of several herpesviruses interfere with PML NB-mediated repression, and in some cases their activities are transferrable between different viruses. For example, the requirement for ICP0 during herpes simplex virus 1 (HSV-1) infection can be largely replaced by ICP0-related proteins expressed by other alphaherpesviruses and even by a combination of the unrelated IE1 and pp71 proteins of human cytomegalovirus (HCMV). Here, we report that ICP0 stimulates gene expression and replication of wt HCMV but cannot replace the need for IE1 during infection by IE1-defective HCMV mutants. Therefore, IE1 includes HCMV-specific functions that cannot be replaced by ICP0.

scholarly journals Histone deacetylase inhibitors reduce the number of herpes simplex virus-1 genomes initiating expression in individual cells

2016 ◽  
Author(s):  
Shapira Lev ◽  
Ralph Maya ◽  
Tomer Enosh ◽  
Cohen Shai ◽  
Kobiler Oren
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Types ◽  
Nuclear Bodies ◽  
Host Proteins ◽  
Herpes Simplex Virus 1 ◽  
Viral Genomes ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

AbstractAlthough many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the infection. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may down regulate the number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.

scholarly journals Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using anIn VitroHigh-Throughput Screening Assay

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Thibaut Deschamps ◽  
Hope Waisner ◽  
Christos Dogrammatzis ◽  
Anuradha Roy ◽  
Shibin Chacko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Screening Assay ◽  
Herpes Simplex Virus 1 ◽  
Ubiquitin Ligase Activity ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes 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-β)-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.IMPORTANCESince 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 infectionin 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.

scholarly journals Mutational pressure by host APOBEC3s more strongly affects genes expressed early in the lytic phase of herpes simplex virus-1 (HSV-1) and human polyomavirus (HPyV) infection

2021 ◽  
Vol 17 (4) ◽  
pp. e1009560
Author(s):  
Maxwell Shapiro ◽  
Laurie T. Krug ◽  
Thomas MacCarthy
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Lytic Cycle ◽  
Human Polyomavirus ◽  
Stochastic Methods ◽  
Herpes Simplex Virus 1 ◽  
Mutational Pressure ◽  
Cytidine Deaminases ◽  
Simplex Virus ◽  
Hsv 1

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later.

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