PARP1 Enhances Influenza A Virus Propagation by Facilitating Degradation of Host Type I Interferon Receptor.

Influenza A virus (IAV) utilizes multiple strategies to confront or evade host type I interferon (IFN)-mediated antiviral responses in order to enhance its own propagation within the host. One such strategy is to induce the degradation of type I IFN receptor 1 (IFNAR1) by utilizing viral hemagglutinin (HA). However, the molecular mechanism behind this process is poorly understood. Here, we report that a cellular protein, poly(ADP-ribose) polymerase 1 (PARP1), plays a critical role in mediating IAV HA-induced degradation of IFNAR1. We identified PARP1 as an interacting partner for IAV HA through mass spectrometry analysis. This interaction was confirmed by coimmunoprecipitation analyses. Furthermore, confocal fluorescence microscopy showed altered localization of endogenous PARP1 upon transient IAV HA expression or during IAV infection. Knockdown or inhibition of PARP1 rescued IFNAR1 levels upon IAV infection or HA expression, exemplifying the importance of PARP1 for IAV-induced reduction of IFNAR1. Notably, PARP1 was crucial for the robust replication of IAV, which was associated with regulation of the type I IFN receptor signaling pathway. These results indicate that PARP1 promotes IAV replication by controlling viral HA-induced degradation of host type I IFN receptor. Altogether, these findings provide novel insight into interactions between influenza virus and the host innate immune response and reveal a new function for PARP1 during influenza virus infection.IMPORTANCE Influenza A virus (IAV) infections cause seasonal and pandemic influenza outbreaks, which pose a devastating global health concern. Despite the availability of antivirals against influenza, new IAV strains continue to persist by overcoming the therapeutics. Therefore, much emphasis in the field is placed on identifying new therapeutic targets that can more effectively control influenza. IAV utilizes several tactics to evade host innate immunity, which include the evasion of antiviral type I interferon (IFN) responses. Degradation of type I IFN receptor (IFNAR) is one known method of subversion, but the molecular mechanism for IFNAR downregulation during IAV infection remains unclear. Here, we have found that a host protein, poly(ADP-ribose) polymerase 1 (PARP1), facilitates IFNAR degradation and accelerates IAV replication. The findings reveal a novel cellular target for the potential development of antivirals against influenza, as well as expand our base of knowledge regarding interactions between influenza and the host innate immunity.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

Casein Kinase 1α Mediates the Degradation of Receptors for Type I and Type II Interferons Caused by Hemagglutinin of Influenza A Virus.

Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus.IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of the type II IFN receptor IFNGR1 and thereby substantially attenuates cellular responses to IFN-γ. Of note, a cellular kinase, casein kinase 1α (CK1α), is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1α is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may provide new insights into the interplay between IAV and host immunity to impact influenza virus pathogenicity.

Sphingosine 1-Phosphate Lyase Enhances the Activation of IKKε To Promote Type I IFN-Mediated Innate Immune Responses to Influenza A Virus Infection.

Sphingosine 1-phosphate (S1P) lyase (SPL) is an intracellular enzyme that mediates the irreversible degradation of the bioactive lipid S1P. We have previously reported that overexpressed SPL displays anti-influenza viral activity; however, the underlying mechanism is incompletely understood. In this study, we demonstrate that SPL functions as a positive regulator of IKKε to propel type I IFN-mediated innate immune responses against viral infection. Exogenous SPL expression inhibited influenza A virus replication, which correlated with an increase in type I IFN production and IFN-stimulated gene accumulation upon infection. In contrast, the lack of SPL expression led to an elevated cellular susceptibility to influenza A virus infection. In support of this, SPL-deficient cells were defective in mounting an effective IFN response when stimulated by influenza viral RNAs. SPL augmented the activation status of IKKε and enhanced the kinase-induced phosphorylation of IRF3 and the synthesis of type I IFNs. However, the S1P degradation-incompetent form of SPL also enhanced IFN responses, suggesting that SPL's pro-IFN function is independent of S1P. Biochemical analyses revealed that SPL, as well as the mutant form of SPL, interacts with IKKε. Importantly, when endogenous IKKε was downregulated using a small interfering RNA approach, SPL's anti-influenza viral activity was markedly suppressed. This indicates that IKKε is crucial for SPL-mediated inhibition of influenza virus replication. Thus, the results illustrate the functional significance of the SPL-IKKε-IFN axis during host innate immunity against viral infection.

For Better or Worse: Modulation of the Host DNA Damage Response by Human Papillomavirus.

High-risk human papillomaviruses (HPVs) are associated with several human cancers. HPVs are small, DNA viruses that rely on host cell machinery for viral replication. The HPV life cycle takes place in the stratified epithelium, which is composed of different cell states, including terminally differentiating cells that are no longer active in the cell cycle. HPVs have evolved mechanisms to persist and replicate in the stratified epithelium by hijacking and modulating cellular pathways, including the DNA damage response (DDR). HPVs activate and exploit DDR pathways to promote viral replication, which in turn increases the susceptibility of the host cell to genomic instability and carcinogenesis. Here, we review recent advances in our understanding of the regulation of the host cell DDR by high-risk HPVs during the viral life cycle and discuss the potential cellular consequences of modulating DDR pathways.

Interplay between the DNA damage response and the life cycle of DNA tumor viruses.

Approximately 20 % of human cancers are associated with virus infection. DNA tumor viruses can induce tumor formation in host cells by disrupting the cell's DNA replication and repair mechanisms. Specifically, these viruses interfere with the host cell's DNA damage response (DDR), which is a complex network of signaling pathways that is essential for maintaining the integrity of the genome. DNA tumor viruses can disrupt these pathways by expressing oncoproteins that mimic or inhibit various DDR components, thereby promoting genomic instability and tumorigenesis. Recent studies have highlighted the molecular mechanisms by which DNA tumor viruses interact with DDR components, as well as the ways in which these interactions contribute to viral replication and tumorigenesis. Understanding the interplay between DNA tumor viruses and the DDR pathway is critical for developing effective strategies to prevent and treat virally associated cancers. In this review, we discuss the current state of knowledge regarding the mechanisms by which human papillomavirus (HPV), merkel cell polyomavirus (MCPyV), Kaposi's sarcoma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV) interfere with DDR pathways to facilitate their respective life cycles, and the consequences of such interference on genomic stability and cancer development.

Chronic LCMV Infection Is Fortified with Versatile Tactics to Suppress Host T Cell Immunity and Establish Viral Persistence.

Ever since the immune regulatory strains of lymphocytic choriomeningitis virus (LCMV), such as Clone 13, were isolated, LCMV infection of mice has served as a valuable model for the mechanistic study of viral immune suppression and virus persistence. The exhaustion of virus-specific T cells was demonstrated during LCMV infection, and the underlying mechanisms have been extensively investigated using LCMV infection in mouse models. In particular, the mechanism for gradual CD8+ T cell exhaustion at molecular and transcriptional levels has been investigated. These studies revealed crucial roles for inhibitory receptors, surface markers, regulatory cytokines, and transcription factors, including PD-1, PSGL-1, CXCR5, and TOX in the regulation of T cells. However, the action mode for CD4+ T cell suppression is largely unknown. Recently, sphingosine kinase 2 was proven to specifically repress CD4+ T cell proliferation and lead to LCMV persistence. As CD4+ T cell regulation was also known to be important for viral persistence, research to uncover the mechanism for CD4+ T cell repression could help us better understand how viruses launch and prolong their persistence. This review summarizes discoveries derived from the study of LCMV in regard to the mechanisms for T cell suppression and approaches for the termination of viral persistence with special emphasis on CD8+ T cells.

Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider.

Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human-animal interfaces in geographical hotspots for emerging infectious diseases. Where human-animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics.

Influenza A virus NS1 induces degradation of sphingosine 1-phosphate lyase to obstruct the host innate immune response.

The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome in order to successfully replicate within the host. We have previously shown that sphingosine 1-phosphate (S1P) lyase (SPL) enhances IKKϵ-mediated type I IFN responses. Here, we demonstrate that the nonstructural protein 1 (NS1) of IAV counteracts the SPL-mediated antiviral response by inducing degradation of SPL. SPL was ubiquitinated and downregulated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit SPL ubiquitination or downregulation. Transiently overexpressed SPL increased phosphorylation of IKKϵ, resulting in enhanced expression of type I IFNs. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.

Sphingosine kinase 2 restricts T cell immunopathology but permits viral persistence.

Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2's role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.

Emerging Connections of S1P-Metabolizing Enzymes with Host Defense and Immunity During Virus Infections.

The sphingosine 1-phosphate (S1P) metabolic pathway is a dynamic regulator of multiple cellular and disease processes. Identification of the immune regulatory role of the sphingosine analog FTY720 led to the development of the first oral therapy for the treatment of an autoimmune disease, multiple sclerosis. Furthermore, inhibitors of sphingosine kinase (SphK), which mediate S1P synthesis, are being evaluated as a therapeutic option for the treatment of cancer. In conjunction with these captivating discoveries, S1P and S1P-metabolizing enzymes have been revealed to display vital functions during virus infections. For example, S1P lyase, which is known for metabolizing S1P, inhibits influenza virus replication by promoting antiviral type I interferon innate immune responses. In addition, both isoforms of sphingosine kinase have been shown to regulate the replication or pathogenicity of many viruses. Pro- or antiviral activities of S1P-metabolizing enzymes appear to be dependent on diverse virus-host interactions and viral pathogenesis. This review places an emphasis on summarizing the functions of S1P-metabolizing enzymes during virus infections and discusses the opportunities for designing pioneering antiviral drugs by targeting these host enzymes.

Transient inhibition of sphingosine kinases confers protection to influenza A virus infected mice.

Influenza continues to pose a threat to public health by causing illness and mortality in humans. Discovering host factors that regulate influenza virus propagation is vital for the development of novel drugs. We have previously reported that sphingosine kinase (SphK) 1 promotes influenza A virus (IAV) replication in vitro. Here we demonstrate that the other isoform of SphK, SphK2 promotes the replication of influenza A virus (IAV) in cultured cells, and temporary inhibition of SphK1 or SphK2 enhances the host defense against influenza in mice. IAV infection led to an increased expression and phosphorylation of SphK2 in host cells. Furthermore, pharmacologic inhibition or siRNA-based knockdown of SphK2 attenuated IAV replication in vitro. Notably, oral administration of an SphK2-specific inhibitor substantially improved the viability of mice following IAV infection. In addition, the local instillation of an SphK1-specific inhibitor or an inhibitor that globally blocks SphK1 and SphK2 provided protection to IAV-infected mice. Collectively, our results indicate that both SphK1 and SphK2 function as proviral factors during IAV infection in vivo. Therefore, SphK1 and SphK2 represent potential host targets for therapeutics against influenza.