Patterns and rates of exonic de novo mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified1,2. To identify further genetic risk factors, we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n= 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant and the overall rate of mutation is only modestly higher than the expected rate. In contrast, there is significantly enriched connectivity among the proteins encoded by genes harboring de novo missense or nonsense mutations, and excess connectivity to prior ASD genes of major effect, suggesting a subset of observed events are relevant to ASD risk. The small increase in rate of de novo events, when taken together with the connections among the proteins themselves and to ASD, are consistent with an important but limited role for de novo point mutations, similar to that documented for de novo copy number variants. Genetic models incorporating these data suggest that the majority of observed de novo events are unconnected to ASD, those that do confer risk are distributed across many genes and are incompletely penetrant (i.e., not necessarily causal). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5 to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favor of CHD8 and KATNAL2 as genuine autism risk factors

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influ-enza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 50-m7G-cappedhost transcripts to prime viral mRNA synthesis (‘‘cap-snatching’’). We hypothesized that start codons withincap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We reportthe existence of this mechanism of gene origination, which we named ‘‘start-snatching.’’ Depending on thereading frame, start-snatching allows the translation of host and viral ‘‘untranslated regions’’ (UTRs) to createN-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show thatboth types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contributeto virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animaland plant viruses, a host-dependent mechanism allows the genesis of hybrid genes

Genomic Approaches Reveal the Interplay of Viral and Cellular Transcription During Infection

Background: Virus:host interactions occur at many levels, including viral entry, viral transcription and replication, and host immune response. Whereas, molecular assays (RT-PCR, northern blot) have been typically used to investigate viral and host transcription. Here we present new next-generation sequencing (NGS) approaches to studying virus:host interactions at the transcriptional level for Influenza and Ebola virus. Aims: Using NGS coupled with custom bioinformatics pipelines, we set out to examine the interplay between host and viral transcription during infection. Focusing on Influenza A virus (IAV), we investigated the substrate preferences for IAV cap-snatching - the process by which the viral RNA-dependent RNA polymerase (RdRP) complex cleaves the first 10-15 nucleotides to prime viral messenger RNA transcription - and its effect on host gene expression and viral protein diversity. In addition, through the application of Gene Co-Expression Network Analysis to a unique sample set of peripheral blood mononuclear cells (PBMC) obtained from Ebola virus-infected patients from Sierra Leone, we explored mechanisms of Ebola pathogenesis and differences in host responses between patients that survived or succumbed to infection. Results: Utilizing a 5’ end “cap-snatch” sequencing technique, CS-Seq, we characterized RNA kinetics throughout infection across cellular compartments. We not only comprehensively profiled viral transcripts but, unlike previous studies, we also quantified the host response at the same time. Analysis of viral mRNAs produced by cap-snatching revealed RdRP’s preference for host transcripts starting with a purine, and the reciprocal avoidance of pyrimidine-rich 5’ TOP genes that play an important role in translation. These nucleotide preferences were conserved amongst other cap-snatching viruses, despite differences in viral polymerases and their subcellular localization. Furthermore, we show that cap-snatching primarily targets short nascent mRNAs (<200nt), as longer transcripts are protected by the cellular Cap Binding Complex. This same mechanism also ensures that viral mRNAs are protected from self-cleavage. We then go on to show that the addition of a short host-oligo increases the probability of introducing a start codon prior to the native start codon, allowing the production of extended viral proteins or out-of-frame products from upstream ORFs (uORFs). The detection of the products of one of these uORFs in mass spectrometry data indicates that the cap-snatching mechanism has the potential to increase viral protein diversity. Because these proteins have not been characterized, they open up new avenues of investigation regarding their role in infectivity. Lastly, we were able to acquire PBMC RNA samples from patients in Sierra Leone’s 2014 Ebola outbreak to study host transcriptional response to infection. By employing gene co-expression network analysis, we were able to observe differential host-immune responses to Ebola infection, correlate them with patient outcome and support them with other OMICs data from the same patients. Conclusions Genomic approaches in the form of sequencing methods and analysis pipelines have provided valuable insight into virus-host interactions that may ultimately result in new targets for drug discover

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3 end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation.

The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response

Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T&nbsp;cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes