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.

Signatures of host-pathogen evolutionary conflict reveal MISTR-A conserved MItochondrial STress Response network.

Host-pathogen conflicts leave genetic signatures in genes that are critical for host defense functions. Using these "molecular scars" as a guide to discover gene functions, we discovered a vertebrate-specific MItochondrial STress Response (MISTR) circuit. MISTR proteins are associated with electron transport chain (ETC) factors and activated by stress signals such as interferon gamma (IFNγ) and hypoxia. Upon stress, ultraconserved microRNAs (miRNAs) down-regulate MISTR1(NDUFA4) followed by replacement with paralogs MItochondrial STress Response AntiViral (MISTRAV) and/or MItochondrial STress Response Hypoxia (MISTRH). While cells lacking MISTR1(NDUFA4) are more sensitive to chemical and viral apoptotic triggers, cells lacking MISTRAV or expressing the squirrelpox virus-encoded vMISTRAV exhibit resistance to the same insults. Rapid evolution signatures across primate genomes for MISTR1(NDUFA4) and MISTRAV indicate recent and ongoing conflicts with pathogens. MISTR homologs are also found in plants, yeasts, a fish virus, and an algal virus indicating ancient origins and suggesting diverse means of altering mitochondrial function under stress. The discovery of MISTR circuitry highlights the use of evolution-guided studies to reveal fundamental biological processes.

Evolutionary Profile for (Host and Viral) MLKL Indicates Its Activities as a Battlefront for Extensive Counteradaptation.

Pathogen infection triggers host innate defenses which may result in the activation of regulated cell death (RCD) pathways such as apoptosis. Given a vital role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptosis. Yet, it is unclear whether components of this emerging RCD pathway display signatures associated with pathogen conflict that are rare in combination but common to key host defense factors, namely, rapid evolution, viral homolog (virolog), and cytokine induction. We leveraged evolutionary sequence analysis that examines rates of amino acid replacement, which revealed: 1) strong and recurrent signatures of positive selection for primate and bat RIPK3 and MLKL, and 2) elevated rates of amino acid substitution on multiple RIPK3/MLKL surfaces suggestive of past antagonism with multiple, distinct pathogen-encoded inhibitors. Furthermore, our phylogenomics analysis across poxvirus genomes illuminated volatile patterns of evolution for a recently described MLKL viral homolog. Specifically, poxviral MLKLs have undergone numerous gene replacements mediated by duplication and deletion events. In addition, MLKL protein expression is stimulated by interferons in human and mouse cells. Thus, MLKL displays all three hallmarks of pivotal immune factors of which only a handful of factors like OAS1 exhibit. These data support the hypothesis that over evolutionary time MLKL functions-which may include execution of necroptosis-have served as a major determinant of infection outcomes despite gene loss in some host genomes.

Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome.

Moebius syndrome (MBS) is a congenital disorder caused by paralysis of the facial and abducens nerves. Although a number of candidate genes have been suspected, so far only mutations in PLXND1 and REV3L are confirmed to cause MBS. Here, we fine mapped the breakpoints of a complex chromosomal rearrangement (CCR) 46,XY,t(7;8;11;13) in a patient with MBS, which revealed 41 clustered breakpoints with typical hallmarks of chromothripsis. Among 12 truncated protein-coding genes, SEMA3A is known to bind to the MBS-associated PLXND1. Intriguingly, the CCR also truncated PIK3CG, which in silico interacts with REVL3 encoded by the other known MBS-gene REV3L, and with the SEMA3A/PLXND1 complex via FLT1. Additional studies of other complex rearrangements may reveal whether the multiple breakpoints in germline chromothripsis may predispose to complex multigenic disorders.

Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape.

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular 'arms races.' Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2'-5'-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.

cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection.

Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.

Germline Chromothripsis Driven by L1-Mediated Retrotransposition and Alu/Alu Homologous Recombination.

Chromothripsis (CTH) is a phenomenon where multiple localized double-stranded DNA breaks result in complex genomic rearrangements. Although the DNA-repair mechanisms involved in CTH have been described, the mechanisms driving the localized "shattering" process remain unclear. High-throughput sequence analysis of a familial germline CTH revealed an inserted SVAE retrotransposon associated with a 110-kb deletion displaying hallmarks of L1-mediated retrotransposition. Our analysis suggests that the SVAE insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1-endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline.

Enrichment of processed pseudogene transcripts in L1-ribonucleoprotein particles.

Long INterspersed Elements (LINE-1s, L1s) are responsible for over one million retrotransposon insertions and 8000 processed pseudogenes (PPs) in the human genome. An active L1 encodes two proteins (ORF1p and ORF2p) that bind with L1 RNA and form L1-ribonucleoprotein particles (RNPs). Although it is believed that the RNA-binding property of ORF1p is critical to recruit other mobile RNAs to the RNP, the identity of recruited RNAs is largely unknown. Here, we used crosslinking and immunoprecipitation followed by deep sequencing to identify RNA components of L1-RNPs. Our results show that in addition to retrotransposed RNAs [L1, Alu and SINE-VNTR-Alu (SVA)], L1-RNPs are enriched with cellular mRNAs, which have PPs in the human genome. Using purified L1-RNPs, we show that PP-source RNAs preferentially serve as ORF2p templates in a reverse transcriptase assay. In addition, we find that exogenous ORF2p binds endogenous ORF1p, allowing reverse transcription of the same PP-source RNAs. These data demonstrate that interaction of a cellular RNA with the L1-RNP is an inside track to PP formation.

Unanticipated loss of inflammasomes in birds.

Inflammasomes are multiprotein complexes that form in response to ligands originating from pathogens as well as alterations of normal cell physiology caused by infection or tissue damage. These structures engage a robust inflammatory immune response that eradicates environmental microbes before they cause disease, and slow the growth of bona fide pathogens. Despite their undeniable utility in immunity, inflammasomes are radically reduced in birds. Perhaps most surprising is that, within all birds, NLRP3 is retained, while its signaling adapter ASC is lost, suggesting that NLRP3 signals via a novel unknown adapter. Crocodilian reptiles and turtles, which share a more recent common ancestor with birds, retain many of the lost inflammasome components, indicating that the deletion of inflammasomes occurred after birds diverged from crocodiles. Some bird lineages have even more extensive inflammasome loss, with songbirds continuing to pare down their inflammasomes until only NLRP3 and CARD8 remain. Remarkably, songbirds have lost caspase-1 but retain the downstream targets of caspase-1: IL-1ß, IL-18, and the YVAD-linker encoding gasdermin A. This suggests that inflammasomes can signal through alternative proteases to activate cytokine maturation and pyroptosis in songbirds. These observations may reveal new contexts of activation that may be relevant to mammalian inflammasomes, and may suggest new avenues of research to uncover the enigmatic nature of the poorly understood NLRP3 inflammasome.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Retrotransposition of marked SVA elements by human L1s in cultured cells.

Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed.

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.

A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins.

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the F ACT- E TS-1 A ntiviral R esponse (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

Virologs, viral mimicry, and virocell metabolism: the expanding scale of cellular functions encoded in the complex genomes of giant viruses.

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses" have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host-referred to here as viral homologs or "virologs"-as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates" immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.

Pathogenic orphan transduction created by a nonreference LINE-1 retrotransposon.

Long INterspersed Element-1 (LINE-1) retrotransposons comprise 17% of the human genome, and move by a potentially mutagenic "copy and paste" mechanism via an RNA intermediate. Recently, the retrotransposition-mediated insertion of a new transcript was described as a novel cause of genetic disease, Duchenne muscular dystrophy, in a Japanese male. The inserted sequence was presumed to derive from a single-copy, noncoding RNA transcribed from chromosome 11q22.3 that retrotransposed into the dystrophin gene. Here, we demonstrate that a nonreference full-length LINE-1 is situated in the proband and maternal genome at chromosome 11q22.3, directly upstream of the sequence, whose copy was inserted into the dystrophin gene. This LINE-1 is highly active in a cell culture assay. LINE-1 insertions are often associated with 3' transduction of adjacent genomic sequences. Thus, the likely explanation for the mutagenic insertion is a LINE-1-mediated 3' transduction with severe 5' truncation. This is the first example of LINE-1-induced human disease caused by an "orphan" 3' transduction.

Exon-trapping mediated by the human retrotransposon SVA.

Although most human retrotransposons are inactive, both inactive and active retrotransposons drive genome evolution and may influence transcription through various mechanisms. In humans, three retrotransposon families are still active, but one of these, SVA, remains mysterious. Here we report the identification of a new subfamily of SVA, which apparently formed after an alternative splicing event where the first exon of the MAST2 gene spliced into an intronic SVA and subsequently retrotransposed. Additional examples of SVA retrotransposing upstream exons due to splicing into SVA were also identified in other primate genomes. After molecular and computational experiments, we found a number of functional 3' splice sites within many different transcribed SVAs across the human and chimpanzee genomes. Using a minigene splicing construct containing an SVA, we observed splicing in cell culture, along with SVA exonization events that introduced premature termination codons (PTCs). These data imply that an SVA residing within an intron in the same orientation as the gene may alter normal gene transcription either by gene-trapping or by introducing PTCs through exonization, possibly creating differences within and across species.

SVA retrotransposons: Evolution and genetic instability.

SINE-VNTR-Alus (SVA) are non-autonomous hominid specific retrotransposons that are associated with disease in humans. SVAs are evolutionarily young and presumably mobilized by the LINE-1 reverse transcriptase in trans. SVAs are currently active and may impact the host through a variety of mechanisms including insertional mutagenesis, exon shuffling, alternative splicing, and the generation of differentially methylated regions (DMR). Here we review SVA biology, including SVA insertions associated with known diseases. Further, we discuss a model describing the initial formation of SVA and the mechanisms by which SVA may impact the host.

Mitochondria and Viral Infection: Advances and Emerging Battlefronts.

Mitochondria are dynamic organelles vital for energy production with now appreciated roles in immune defense. During microbial infection, mitochondria serve as signaling hubs to induce immune responses to counteract invading pathogens like viruses. Mitochondrial functions are central to a variety of antiviral responses including apoptosis and type I interferon signaling (IFN-I). While apoptosis and IFN-I mediated by mitochondrial antiviral signaling (MAVS) are well-established defenses, new dimensions of mitochondrial biology are emerging as battlefronts during viral infection. Increasingly, it has become apparent that mitochondria serve as reservoirs for distinct cues that trigger immune responses and that alterations in mitochondrial morphology may also tip infection outcomes. Furthermore, new data are foreshadowing pivotal roles for classic, homeostatic facets of this organelle as host-virus interfaces, namely, the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) complexes like respiratory supercomplexes. Underscoring the importance of "housekeeping" mitochondrial activities in viral infection is the growing list of viral-encoded inhibitors including mimics derived from cellular genes that antagonize these functions. For example, virologs for ETC factors and several enzymes from the TCA cycle have been recently identified in DNA virus genomes and serve to pinpoint new vulnerabilities during infection. Here, we highlight recent advances for known antiviral functions associated with mitochondria as well as where the next battlegrounds may be based on viral effectors. Collectively, new methodology and mechanistic insights over the coming years will strengthen our understanding of how an ancient molecular truce continues to defend cells against viruses.

Effects of cis and trans genetic ancestry on gene expression in African Americans.

Variation in gene expression is a fundamental aspect of human phenotypic variation. Several recent studies have analyzed gene expression levels in populations of different continental ancestry and reported population differences at a large number of genes. However, these differences could largely be due to non-genetic (e.g., environmental) effects. Here, we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that individuals from this population inherit variable proportions of two continental ancestries. We first relate gene expression levels in individual African Americans to their genome-wide proportion of European ancestry. The results provide strong evidence of a genetic contribution to expression differences between European and African populations, validating previous findings. Second, we infer local ancestry (0, 1, or 2 European chromosomes) at each location in the genome and investigate the effects of ancestry proximal to the expressed gene (cis) versus ancestry elsewhere in the genome (trans). Both effects are highly significant, and we estimate that 12+/-3% of all heritable variation in human gene expression is due to cis variants.