Autophagic degradation of PML promotes susceptibility to HSV-1 by stress-induced corticosterone.

Rationale: Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that can cause a variety of clinical syndromes including mucocutaneous disease and HSV-1 encephalitis (HSE). Here, we characterize the molecular mechanisms underlying the susceptibility to HSV-1 under stressful conditions. Methods: Restraint stress and corticosterone (CORT, a primary stress hormone) were respectively used to establish HSV-1 susceptible model in vivo and in vitro. Viral titers were determined by plaque assay. Western blotting, immunofluorescence, transmission electron microscopy (TEM), qRT-PCR, H&E staining, IHC staining and flow cytometry were employed to evaluate virus-related protein expressions and detect the activation of autophagy. Loss- and gain-function assays, co-immunoprecipitation (co-IP) technique and autophagy agonist/antagonist treatments were applied in mechanistic experiments. Results: Restraint stress increased the susceptibility of mouse brain to HSV-1. Similarly, CORT treatment enhanced the susceptibility of neural cells to HSV-1. Furthermore, PML protein level in HSV-1 infected brain tissues and neural cells was remarkably decreased by stress treatment in vivo or CORT treatment in vitro, while its transcriptional level was not affected. Notably, a striking decline in protein expressions of ICP27 and gB was observed in PML-overexpressing cells, which was reversed by CORT treatment. By contrast, protein expression of gB was increased by knockdown with si-PML in virus-infected SH-SY5Y cells. We further discovered that CORT-driven PML degradation was dependent on the activation of autophagy in a ULK1-independent manner, rather than proteasome pathway. Bafilomycin A1 (BaF1) attenuated the augmentation effect of CORT on HSV-1 infection. The expressions of viral proteins were reduced in LC3-depleted cells, and the degradation of PML by CORT-induced autophagy was prevented in cells with LC3 knockdown by RNAi. Interestingly, PML was revealed to interact with the autophagic cargo receptor P62 and the autophagic effector protein LC3. Additionally, CORT failed to increase gB protein level when PML was silenced, providing direct evidence linking autophagic degradation of PML and CORT-induced virus susceptibility. Conclusion: Our results revealed that restraint stress/CORT increased HSV-1 susceptibility by delivering PML into autolysosomes for degradation. The results obtained from in vitro and in vivo models not only demonstrated the adverse effects of stress on HSV-1 infection, but also systematically investigated the underlying molecular mechanisms. These discoveries broaden our understanding of the interplay between host and viruses, and a comprehensive understanding of the role of autophagy in viral infection will provide information for future development of innovative drugs against viral infection.

Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1.

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.

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.

Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance.

Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, forms large aggregate-like assemblies visualized as nuclear foci. Here we demonstrate that Aire utilizes its caspase activation recruitment domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization also makes Aire susceptible to interaction with promyelocytic leukemia protein (PML) bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs the transcriptional activity of Aire, while dispersing PML bodies with a viral antagonist restores this activity. Our study thus reveals a new regulatory role of PML bodies in Aire function, and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated protein quality control.

Diverse genetic-driven immune landscapes dictate tumor progression through distinct mechanisms.

Multiple immune-cell types can infiltrate tumors and promote progression and metastasis through different mechanisms, including immunosuppression. How distinct genetic alterations in tumors affect the composition of the immune landscape is currently unclear. Here, we characterized the immune-cell composition of prostate cancers driven by the loss of the critical tumor suppressor gene Pten, either alone or in combination with the loss of Trp53, Zbtb7a or Pml. We observed a striking quantitative and qualitative heterogeneity that was directly dependent on the specific genetic events in the tumor and ranged from 'cold', noninflamed tumors to massively infiltrated landscapes-results with important therapeutic implications. Further, we showed these qualitative differences in transcriptomic analysis of human prostate cancer samples. These data suggest that patient stratification on the basis of integrated genotypic-immunophenotypic analyses may be necessary for successful clinical trials and tailored precision immunological therapies.

Nuclear domain 10 components upregulated via interferon during human cytomegalovirus infection potently regulate viral infection.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-ß neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-ß pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-ß in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-ß.