CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses.

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.

Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages.

Even though gammaherpesvirus and parasitic infections are endemic in parts of the world, there is a lack of understanding about the outcome of coinfection. In humans, coinfections usually occur sequentially, with fluctuating order and timing in different hosts. However, experimental studies in mice generally do not address the variables of order and timing of coinfections. We sought to examine the variable of coinfection order in a system of gammaherpesvirus-helminth coinfection. Our previous work demonstrated that infection with the intestinal parasite, Heligmosomoides polygyrus, induced transient reactivation from latency of murine gammaherpesvirus-68 (MHV68). In this report, we reverse the order of coinfection, infecting with H. polygyrus first, followed by MHV68, and examined the effects of preexisting parasite infection on MHV68 acute and latent infection. We found that preexisting parasite infection increased the propensity of MHV68 to reactivate from latency. However, when we examined the mechanism for reactivation, we found that preexisting parasite infection increased the ability of MHV68 to reactivate in a vitamin A dependent manner, a distinct mechanism to what we found previously with parasite-induced reactivation after latency establishment. We determined that H. polygyrus infection increased both acute and latent MHV68 infection in a population of tissue resident macrophages, called large peritoneal macrophages. We demonstrate that this population of macrophages and vitamin A are required for increased acute and latent infection during parasite coinfection.

Norovirus MLKL-like protein initiates cell death to induce viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.

Norovirus MLKL-like pore forming protein initiates programed cell death for viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but a mechanism of norovirus-infection triggered cell death and lysis are unknown. Here we have identified a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase contains a N-terminal four helix bundle domain homologous to the pore forming domain of the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL). Norovirus NTPase acquired a mitochondrial localization signal, thereby inducing cell death by targeting mitochondria. NTPase full length (NTPase-FL) and N-terminal fragment (NTPase-NT) bound mitochondrial membrane lipid cardiolipin, permeabilized mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NTPase were essential for cell death, virus egress from cells and virus replication in mice. These findings suggest that noroviruses stole a MLKL-like pore forming domain and co-opted it to facilitate viral egress by inducing mitochondrial dysfunction.

WY14643 Increases Herpesvirus Replication and Inhibits IFNß Production Independently of PPARα Expression.

Peroxisome proliferator activated receptor (PPAR) agonists are commonly used to treat metabolic disorders in humans because they regulate fatty acid oxidation and cholesterol metabolism. In addition to their roles in controlling metabolism, PPAR agonists also regulate inflammation and are immunosuppressive in models of autoimmunity. We aimed to test whether activation of PPARα with clinically relevant ligands could impact gammaherpesvirus infection using murine gammaherpesvirus-68 (MHV68, MuHV-4). We found that PPAR agonists WY14643 and fenofibrate increased herpesvirus replication in vitro. In vivo, WY14643 increased viral replication and caused lethality in mice. Unexpectedly, these effects proved independent of PPARα. We found that WY14643 suppressed production of type I interferon after MHV68 infection in vitro and in vivo. Taken together, our data indicate that caution should be employed when using PPARα agonists in immuno-metabolic studies, as they can have off-target effects on viral replication through the inhibition of type I interferon production. IMPORTANCE PPAR agonists are used clinically to treat both metabolic and inflammatory disorders. Because viruses are known to rewire host metabolism to their own benefit, the intersection of immunity, metabolism, and virology is an important research area. Our article is an important contribution to this field for two reasons. First, it shows a role for PPARα agonists in altering virus replication. Second, it shows that PPARα agonists can affect virus replication in a manner independent of their predicted target. This knowledge is valuable for anyone seeking to use PPARα agonists as a research tool.

Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.

A diverse group of antimicrobial proteins (AMPs) helps protect the mammalian intestine from varied microbial challenges. We show that small proline-rich protein 2A (SPRR2A) is an intestinal antibacterial protein that is phylogenetically unrelated to previously discovered mammalian AMPs. In this study, SPRR2A was expressed in Paneth cells and goblet cells and selectively killed Gram-positive bacteria by disrupting their membranes. SPRR2A shaped intestinal microbiota composition, restricted bacterial association with the intestinal surface, and protected against Listeria monocytogenes infection. SPRR2A differed from other intestinal AMPs in that it was induced by type 2 cytokines produced during helminth infection. Moreover, SPRR2A protected against helminth-induced bacterial invasion of intestinal tissue. Thus, SPRR2A is a distinctive AMP triggered by type 2 immunity that protects the intestinal barrier during helminth infection.

Helminth virus co-infection: Implications for women's health.

In many parts of the world, women are co-infected with intestinal helminths and sexually transmitted pathogens. In this issue of Cell Host & Microbe, Chetty et al. demonstrate that intestinal helminth infection increases epithelial damage and pathology associated with herpes virus infection.

Th2 Cytokine Modulates Herpesvirus Reactivation in a Cell Type Specific Manner.

Gammaherpesviruses, such as Epstein-Barr virus (EBV), Kaposi's sarcoma associated virus (KSHV), and murine γ-herpesvirus 68 (MHV68), establish latent infection in B cells, macrophages, and non-lymphoid cells, and can induce both lymphoid and non-lymphoid cancers. Research on these viruses has relied heavily on immortalized B cell and endothelial cell lines. Therefore, we know very little about the cell type specific regulation of virus infection. We have previously shown that treatment of MHV68-infected macrophages with the cytokine interleukin-4 (IL-4) or challenge of MHV68-infected mice with an IL-4-inducing parasite leads to virus reactivation. However, we do not know if all latent reservoirs of the virus, including B cells, reactivate the virus in response to IL-4. Here we used an in vivo approach to address the question of whether all latently infected cell types reactivate MHV68 in response to a particular stimulus. We found that IL-4 receptor expression on macrophages was required for IL-4 to induce virus reactivation, but that it was dispensable on B cells. We further demonstrated that the transcription factor, STAT6, which is downstream of the IL-4 receptor and binds virus gene 50 N4/N5 promoter in macrophages, did not bind to the virus gene 50 N4/N5 promoter in B cells. These data suggest that stimuli that promote herpesvirus reactivation may only affect latent virus in particular cell types, but not in others.Importance Herpesviruses establish life-long quiescent infections in specific cells in the body, and only reactivate to produce infectious virus when precise signals induce them to do so. The signals that induce herpesvirus reactivation are often studied only in one particular cell type infected with the virus. However, herpesviruses establish latency in multiple cell types in their hosts. Using murine gammaherpesvirus-68 (MHV68) and conditional knockout mice, we examined the cell type specificity of a particular reactivation signal, interleukin-4 (IL-4). We found that IL-4 only induced herpesvirus reactivation from macrophages, but not from B cells. This work indicates that regulation of virus latency and reactivation is cell type specific. This has important implications for therapies aimed at either promoting or inhibiting reactivation for the control or elimination of chronic viral infections.

Reactive oxygen species oxidize STING and suppress interferon production.

Reactive oxygen species (ROS) are by-products of cellular respiration that can promote oxidative stress and damage cellular proteins and lipids. One canonical role of ROS is to defend the cell against invading bacterial and viral pathogens. Curiously, some viruses, including herpesviruses, thrive despite the induction of ROS, suggesting that ROS are beneficial for the virus. However, the underlying mechanisms remain unclear. Here, we found that ROS impaired interferon response during murine herpesvirus infection and that the inhibition occurred downstream of cytoplasmic DNA sensing. We further demonstrated that ROS suppressed the type I interferon response by oxidizing Cysteine 147 on murine stimulator of interferon genes (STING), an ER-associated protein that mediates interferon response after cytoplasmic DNA sensing. This inhibited STING polymerization and activation of downstream signaling events. These data indicate that redox regulation of Cysteine 147 of mouse STING, which is equivalent to Cysteine 148 of human STING, controls interferon production. Together, our findings reveal that ROS orchestrates anti-viral immune responses, which can be exploited by viruses to evade cellular defenses.

Microbial Sensing by Intestinal Myeloid Cells Controls Carcinogenesis and Epithelial Differentiation.

Physiologic microbe-host interactions in the intestine require the maintenance of the microbiota in a luminal compartment through a complex interplay between epithelial and immune cells. However, the roles of mucosal myeloid cells in this process remain incompletely understood. In this study, we identified that decreased myeloid cell phagocytic activity promotes colon tumorigenesis. We show that this is due to bacterial accumulation in the lamina propria and present evidence that the underlying mechanism is bacterial induction of prostaglandin production by myeloid cells. Moreover, we show that similar events in the normal colonic mucosa lead to reductions in Tuft cells, goblet cells, and the mucus barrier of the colonic epithelium. These alterations are again linked to the induction of prostaglandin production in response to bacterial penetration of the mucosa. Altogether, our work highlights immune cell-epithelial cell interactions triggered by the microbiota that control intestinal immunity, epithelial differentiation, and carcinogenesis.

Making Mouse Models That Reflect Human Immune Responses.

Humans are infected with a variety of acute and chronic pathogens over the course of their lives, and pathogen-driven selection has shaped the immune system of humans. The same is likely true for mice. However, laboratory mice we use for most biomedical studies are bred in ultra-hygienic environments, and are kept free of specific pathogens. We review recent studies that indicate that pathogen infections are important for the basal level of activation and the function of the immune system. Consideration of these environmental exposures of both humans and mice can potentially improve mouse models of human disease.

Sequential Infection with Common Pathogens Promotes Human-like Immune Gene Expression and Altered Vaccine Response.

Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans.

Coinfections: Another Variable in the Herpesvirus Latency-Reactivation Dynamic.

Chronic viruses, such as herpesviruses, shape host physiology. These viruses modulate the inflammatory state of the immune system and have evolved to harness inflammation as a mechanism to regulate viral latency and reactivation. In this review, I examine some of the recent work demonstrating the important role of inflammation in the regulation of the herpesvirus life cycle and discuss recent work that implicates coinfection in the regulation of herpesvirus latency.

Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation.

Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency.

Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection.

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.

Pervasive transcription of a herpesvirus genome generates functionally important RNAs.

Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi's sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely "noise" generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.

Latent gammaherpesvirus 68 infection induces distinct transcriptional changes in different organs.

Previous studies identified a role for latent herpesvirus infection in cross-protection against infection and exacerbation of chronic inflammatory diseases. Here, we identified more than 500 genes differentially expressed in spleens, livers, or brains of mice latently infected with gammaherpesvirus 68 and found that distinct sets of genes linked to different pathways were altered in the spleen compared to those in the liver. Several of the most differentially expressed latency-specific genes (e.g., the gamma interferon [IFN-γ], Cxcl9, and Ccl5 genes) are associated with known latency-specific phenotypes. Chronic herpesvirus infection, therefore, significantly alters the transcriptional status of host organs. We speculate that such changes may influence host physiology, the status of the immune system, and disease susceptibility.

Identification of novel microRNA-like molecules generated from herpesvirus and host tRNA transcripts.

We applied deep sequencing technology to small RNA fractions from cells lytically infected with murine gammaherpesvirus 68 (gammaHV68) in order to define in detail small RNAs generated from a cluster of tRNA-related polycistronic structures located at the left end of the viral genome. We detected 10 new candidate microRNAs (miRNAs), six of which were confirmed by Northern blot analysis, leaving four as provisional. In addition, we determined that previously identified and annotated viral miRNA molecules were not necessarily represented as the most abundant sequence originating from a transcript. Based on these new small RNAs and previously reported gammaHV68 miRNAs, we were able to further describe and annotate the distinctive gammaHV68 tRNA-miRNA structures. We used this deep sequencing data and computational analysis to identify similar structures in the mouse genome and validated that these host structures also give rise to small RNAs. This reveals a possible convergent usage of tRNA/polymerase III (pol III) transcripts to generate small RNAs from both mammalian and viral genomes.

Latent herpesvirus infection arms NK cells.

Natural killer (NK) cells were identified by their ability to kill target cells without previous sensitization. However, without an antecedent "arming" event, NK cells can recognize, but are not equipped to kill, target cells. How NK cells become armed in vivo in healthy hosts is unclear. Because latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that latent herpesvirus infection would arm NK cells. Here we show that NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. NK-cell arming occurred rapidly in the latently infected host and did not require acute viral infection. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge. Thus, the immune environment created by latent herpesvirus infection provides a mechanism whereby host NK-cell function is enhanced in vivo.