Immune- and Nonimmune-Compartment-Specific Interferon Responses Are Critical Determinants of Herpes Simplex Virus-Induced Generalized Infections and Acute Liver Failure.

The interferon (IFN) response to viral pathogens is critical for host survival. In humans and mouse models, defects in IFN responses can result in lethal herpes simplex virus 1 (HSV-1) infections, usually from encephalitis. Although rare, HSV-1 can also cause fulminant hepatic failure, which is often fatal. Although herpes simplex encephalitis has been extensively studied, HSV-1 generalized infections and subsequent acute liver failure are less well understood. We previously demonstrated that IFN-αßγR-/- mice are exquisitely susceptible to liver infection following corneal infection with HSV-1. In this study, we used bone marrow chimeras of IFN-αßγR-/- (AG129) and wild-type (WT; 129SvEv) mice to probe the underlying IFN-dependent mechanisms that control HSV-1 pathogenesis. After infection, WT mice with either IFN-αßγR-/- or WT marrow exhibited comparable survival, while IFN-αßγR-/- mice with WT marrow had a significant survival advantage over their counterparts with IFN-αßγR-/- marrow. Furthermore, using bioluminescent imaging to maximize data acquisition, we showed that the transfer of IFN-competent hematopoietic cells controlled HSV-1 replication and damage in the livers of IFN-αßγR-/- mice. Consistent with this, the inability of IFN-αßγR-/- immune cells to control liver infection in IFN-αßγR-/- mice manifested as profoundly elevated aspartate transaminase (AST) and alanine transaminase (ALT) levels, indicative of severe liver damage. In contrast, IFN-αßγR-/- mice receiving WT marrow exhibited only modest elevations of AST and ALT levels. These studies indicate that IFN responsiveness of the immune system is a major determinant of viral tropism and damage during visceral HSV infections. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infection is an incurable viral infection with the most significant morbidity and mortality occurring in neonates and patients with compromised immune systems. Severe pathologies from HSV include the blindness-inducing herpetic stromal keratitis, highly debilitating and lethal herpes simplex encephalitis, and generalized infections that can lead to herpes simplex virus-induced acute liver failure. While immune compromise is a known factor, the precise mechanisms that lead to generalized HSV infections are unknown. In this study, we used and developed a mouse model system in combination with real-time bioluminescence imaging to demonstrate the relative importance of the immune and nonimmune compartments for containing viral spread and promoting host survival after corneal infection. Our results shed light on the pathogenesis of HSV infections that lead to generalized infection and acute liver failure.

Role of the DNA Sensor STING in Protection from Lethal Infection following Corneal and Intracerebral Challenge with Herpes Simplex Virus 1.

UNLABELLED: STING is a protein in the cytosolic DNA and cyclic dinucleotide sensor pathway that is critical for the initiation of innate responses to infection by various pathogens. Consistent with this, herpes simplex virus 1 (HSV-1) causes invariable and rapid lethality in STING-deficient (STING(-/-)) mice following intravenous (i.v.) infection. In this study, using real-time bioluminescence imaging and virological assays, as expected, we demonstrated that STING(-/-) mice support greater replication and spread in ocular tissues and the nervous system. In contrast, they did not succumb to challenge via the corneal route even with high titers of a virus that was routinely lethal to STING(-/-) mice by the i.v. route. Corneally infected STING(-/-) mice also showed increased periocular disease and increased corneal and trigeminal ganglia titers, although there was no difference in brain titers. They also showed elevated expression of tumor necrosis factor alpha (TNF-α) and CXCL9 relative to control mice but surprisingly modest changes in type I interferon expression. Finally, we also showed that HSV strains lacking the ability to counter autophagy and the PKR-driven antiviral state had near-wild-type virulence following intracerebral infection of STING(-/-) mice. Together, these data show that while STING is an important component of host resistance to HSV in the cornea, its previously shown immutable role in mediating host survival by the i.v. route was not recapitulated following a mucosal infection route. Furthermore, our data are consistent with the idea that HSV counters STING-mediated induction of the antiviral state and autophagy response, both of which are critical factors for survival following direct infection of the nervous system. IMPORTANCE: HSV infections represent an incurable source of morbidity and mortality in humans and are especially severe in neonatal and immunocompromised populations. A key step in the development of an immune response is the recognition of microbial components within infected cells. The host protein STING is important in this regard for the recognition of HSV DNA and the subsequent triggering of innate responses. STING was previously shown to be essential for protection against lethal challenge from intravenous HSV-1 infection. In this study, we show that the requirement for STING depends on the infection route. In addition, STING is important for appropriate regulation of the inflammatory response in the cornea, and our data are consistent with the idea that HSV modulates STING activity through inhibition of autophagy. Our results elucidate the importance of STING in host protection from HSV-1 and demonstrate the redundancy of host protective mechanisms, especially following mucosal infection.

ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity.

Activation of the RIG-I-like receptors, retinoic-acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15, the mechanistic roles of which in innate immunity still remain enigmatic. In the present study, we report that ISG15 conjugation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization and thereby triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease of SARS-CoV-2, a recently emerged coronavirus that has caused the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a key immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.

ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease.

Activation of the RIG-I-like receptors, RIG-I and MDA5, establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15 whose mechanistic roles in innate immunity still remain enigmatic. Here we report that ISGylation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISG15 conjugation to the caspase activation and recruitment domains of MDA5 promotes the formation of higher-order assemblies of MDA5 and thereby triggers activation of innate immunity against a range of viruses including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease (PLpro) of SARS-CoV-2, a recently emerged coronavirus that causes the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a novel immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.

TRIM23 mediates virus-induced autophagy via activation of TBK1.

Autophagy and interferon (IFN)-mediated innate immunity are critical antiviral defence mechanisms, and recent evidence indicated that tripartite motif (TRIM) proteins are important regulators of both processes. Although the role of TRIM proteins in modulating antiviral cytokine responses has been well established, much less is known about their involvement in autophagy in response to different viral pathogens. Through a targeted RNAi screen examining the relevance of selected TRIM proteins in autophagy induced by herpes simplex virus 1 (HSV-1), encephalomyocarditis virus (EMCV) and influenza A virus (IAV), we identified several TRIM proteins that regulate autophagy in a virus-species-specific manner, as well as a few TRIM proteins that were essential for autophagy triggered by all three viruses and rapamycin, among them TRIM23. TRIM23 was critical for autophagy-mediated restriction of multiple viruses, and this activity was dependent on both its RING E3 ligase and ADP-ribosylation factor (ARF) GTPase activity. Mechanistic studies revealed that unconventional K27-linked auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23 and activation of TANK-binding kinase 1 (TBK1) by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor p62. Our work identifies the TRIM23-TBK1-p62 axis as a key component of selective autophagy and further reveals a role for K27-linked ubiquitination in GTPase-dependent TBK1 activation.

The differential interferon responses of two strains of Stat1-deficient mice do not alter susceptibility to HSV-1 and VSV in vivo.

Stat1 is a pivotal transcription factor for generation of the interferon (IFN)-dependent antiviral response. Two Stat1 knockout mouse lines have been previously generated, one deleted the N-terminal domain (ΔNTD) and one in the DNA-binding domain (ΔDBD). These widely-used strains are assumed interchangeable, and both are highly susceptible to various pathogens. In this study, primary cells derived from ΔNTD mice were shown to be significantly more responsive to IFN, and established an antiviral state with greater efficiency than cells derived from ΔDBD mice, following infection with vesicular stomatitis virus and herpes simplex virus type-1. Also, while mice from both strains succumbed rapidly and equally to virus infection, ΔDBD mice supported significantly higher replication in brains and livers than ΔNTD mice. Endpoint-type experimental comparisons of these mouse strains are therefore misleading in failing to indicate important differences in virus replication and innate response.

Synergistic control of herpes simplex virus pathogenesis by IRF-3, and IRF-7 revealed through non-invasive bioluminescence imaging.

Interferon regulatory factors IRF-3 and IRF-7 are central to the establishment of the innate antiviral response. This study examines HSV-1 pathogenesis in IRF-3(-/-), IRF-7(-/-) and double-deleted IRF3/7(-/-) (DKO) mice. Bioluminescence imaging of infection revealed that DKO mice developed visceral infection following corneal inoculation, along with increased viral burdens in all tissues relative to single knockout mice. While all DKO mice synchronously reached endpoint criteria 5 days post infection, the IRF-7(-/-) mice survived longer, indicating that although IRF-7 is dominant, IRF-3 also plays a role in controlling disease. Higher levels of systemic pro-inflammatory cytokines were found in IRF7(-/-) and DKO mice relative to wild-type and IRF-3(-/-) mice, and IL-6 and G-CSF, indicative of sepsis, were increased in the DKO mice relative to wild-type or single-knockout mice. In addition to controlling viral replication, IRF-3 and -7 therefore play coordinating roles in modulation of inflammation during HSV infection.

Elevated levels of the norspermidine synthesis enzyme NspC enhance Vibrio cholerae biofilm formation without affecting intracellular norspermidine concentrations.

Biofilm formation in Vibrio cholerae is in part regulated by norspermidine, a polyamine synthesized by the enzyme carboxynorspermidine decarboxylase (NspC). The absence of norspermidine in the cell leads to a marked reduction in V. cholerae biofilm formation by an unknown mechanism. In this work, we show that overexpression of nspC results in large increases in biofilm formation and vps gene expression as well as a significant decrease in motility. Interestingly, increased NspC levels do not lead to increased concentrations of norspermidine in the cell. Our results show that NspC levels inversely regulate biofilm and motility and implicate the presence of an effective feedback mechanism maintaining norspermidine homeostasis in V. cholerae. Moreover, we provide evidence that NspC and the norspermidine sensor protein, NspS, provide independent and distinct inputs into the biofilm regulatory network.

Bioluminescent imaging reveals divergent viral pathogenesis in two strains of Stat1-deficient mice, and in αßγ interferon receptor-deficient mice.

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1(-/-) and IFNαßγR(-/-) mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1(-/-) mice and in IFNαßγR(-/-) mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1(-/-) (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNαßγR(-/-) mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1(-/-) and IFNαßγR(-/-) mice, we infected an additional Stat1(-/-) strain deleted in the DNA-binding domain (129Stat1(-/-) (DBD)). These 129Stat1(-/-) (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNαßγR(-/-) mice. This lethal pattern was also observed when 129Stat1(-/-) (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1(-/-) (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1(-/-) mouse strains. The data are consistent with the hypothesis that Stat1(-/-) (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons between knockout mouse strains in viral pathogenesis, and may also be relevant to the causation of HSV hepatitis in humans, a rare but frequently fatal infection.

Spermidine regulates Vibrio cholerae biofilm formation via transport and signaling pathways.

Vibrio cholerae, the causative agent of the devastating diarrheal disease cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural environment and in the human host. Development of V. cholerae biofilms are regulated by complex regulatory networks that respond to environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. cholerae. We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli. We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. cholerae via multiple pathways involving both transport and signaling networks.