Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we ...describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous
genes but instead carries the human
locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.
The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by ...extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35–45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCPs) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name “Mriyaviricetes” (after Ukrainian “mriya,” dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family “Gamadviridae.” The previously characterized members of these families, yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism.IMPORTANCEThe origin of giant viruses of eukaryotes that belong to the phylum Nucleocytoviricota is not thoroughly understood and remains a matter of major interest and debate. Here, we combine metagenome database searches with extensive protein sequence and structure analysis to describe a distinct group of viruses with comparatively small genomes of 35–45 kilobases that appear to comprise a distinct class within the phylum Nucleocytoviricota that we provisionally named “Mriyaviricetes.” Mriyaviruses appear to be the closest identified relatives of the ancestors of the Nucleocytoviricota. Analysis of proteins encoded in mriyavirus genomes suggests that they replicate their genome via the rolling circle mechanism that is unusual among viruses with double-stranded DNA genomes and so far not described for members of Nucleocytoviricota.
Interferon (IFN) activates the transcription of several hundred of IFN stimulated genes (ISGs) that constitute a highly effective antiviral defense program. Cell-to-cell variability in the induction ...of ISGs is well documented, but its source and effects are not completely understood. The molecular mechanisms behind this heterogeneity have been related to randomness in molecular events taking place during the JAK-STAT signaling pathway. Here, we study the sources of variability in the induction of the IFN-alpha response by using MxA and IFIT1 activation as read-out. To this end, we integrate time-resolved flow cytometry data and stochastic modeling of the JAK-STAT signaling pathway. The complexity of the IFN response was matched by fitting probability distributions to time-course flow cytometry snapshots. Both, experimental data and simulations confirmed that the MxA and IFIT1 induction circuits generate graded responses rather than all-or-none responses. Subsequently, we quantify the size of the intrinsic variability at different steps in the pathway. We found that stochastic effects are transiently strong during the ligand-receptor activation steps and the formation of the ISGF3 complex, but negligible for the final induction of the studied ISGs. We conclude that the JAK-STAT signaling pathway is a robust biological circuit that efficiently transmits information under stochastic environments.
Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of ...viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.
Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing ...potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response.
We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction.
HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication.
Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence.
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As obligate parasites, viruses strictly depend on host cell translation for the production of new progeny, yet infected cells also synthesize antiviral proteins to limit virus infection. Modulation ...of host cell translation therefore represents a frequent strategy by which viruses optimize their replication and spread. Here we sought to define how host cell translation is regulated during infection of human cells with dengue virus (DENV) and Zika virus (ZIKV), two positive-strand RNA flaviviruses. Polysome profiling and analysis of de novo protein synthesis revealed that flavivirus infection causes potent repression of host cell translation, while synthesis of viral proteins remains efficient. Selective repression of host cell translation was mediated by the DENV polyprotein at the level of translation initiation. In addition, DENV and ZIKV infection suppressed host cell stress responses such as the formation of stress granules and phosphorylation of the translation initiation factor eIF2α (α subunit of eukaryotic initiation factor 2). Mechanistic analyses revealed that translation repression was uncoupled from the disruption of stress granule formation and eIF2α signaling. Rather, DENV infection induced p38-Mnk1 signaling that resulted in the phosphorylation of the eukaryotic translation initiation factor eIF4E and was essential for the efficient production of virus particles. Together, these results identify the uncoupling of translation suppression from the cellular stress responses as a conserved strategy by which flaviviruses ensure efficient replication in human cells.
For efficient production of new progeny, viruses need to balance their dependency on the host cell translation machinery with potentially adverse effects of antiviral proteins produced by the infected cell. To achieve this, many viruses evolved mechanisms to manipulate host cell translation. Here we find that infection of human cells with two major human pathogens, dengue virus (DENV) and Zika virus (ZIKV), leads to the potent repression of host cell translation initiation, while the synthesis of viral protein remains unaffected. Unlike other RNA viruses, these flaviviruses concomitantly suppress host cell stress responses, thereby uncoupling translation suppression from stress granule formation. We identified that the p38-Mnk1 cascade regulating phosphorylation of eIF4E is a target of DENV infection and plays an important role in virus production. Our results define several molecular interfaces by which flaviviruses hijack host cell translation and interfere with stress responses to optimize the production of new virus particles.
Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through ...significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds.
Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.
Bacteriophages play key roles in the dynamics of the human microbiome. By far the most abundant components of the human gut virome are tailed bacteriophages of the realm Duplodnaviria, in particular, ...crAss-like phages. However, apart from duplodnaviruses, the gut virome has not been dissected in detail. Here we report a comprehensive census of a minor component of the gut virome, the tailless bacteriophages of the realm Varidnaviria. Tailless phages are primarily represented in the gut by prophages, that are mostly integrated in genomes of Alphaproteobacteria and Verrucomicrobia and belong to the order Vinavirales, which currently consists of the families Corticoviridae and Autolykiviridae. Phylogenetic analysis of the major capsid proteins (MCP) suggests that at least three new families should be established within Vinavirales to accommodate the diversity of prophages from the human gut virome. Previously, only the MCP and packaging ATPase genes were reported as conserved core genes of Vinavirales. Here we report an extended core set of 12 proteins, including MCP, packaging ATPase, and previously undetected lysis enzymes, that are shared by most of these viruses. We further demonstrate that replication system components are frequently replaced in the genomes of Vinavirales, suggestive of selective pressure for escape from yet unknown host defenses or avoidance of incompatibility with coinfecting related viruses. The results of this analysis show that, in a sharp contrast to marine viromes, varidnaviruses are a minor component of the human gut virome. Moreover, they are primarily represented by prophages, as indicated by the analysis of the flanking genes, suggesting that there are few, if any, lytic varidnavirus infections in the gut at any given time. These findings complement the existing knowledge of the human gut virome by exploring a group of viruses that has been virtually overlooked in previous work.
HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA pool and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by ...Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting the conjugation of Nedd8 to Cullin-RING ubiquitin ligases impairs degradation of substrates, restoring SMC5/6 levels. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants.
An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA and integrant-driven expression.
siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in 3 infection models. Using the HBV inducible cell line HepAD38 as a model we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment.
Neddylation is required both for the formation of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B.
While there are effective treatments for chronic hepatitis B, they are rarely able to induce functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.
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•Neddylation plays a dual role in HBV expression from viral integrants and episomal cccDNA.•Impaired neddylation suppresses production of HBsAg expressed from viral integrants.•Neddylation promotes HBsAg generation from viral integrants in an HBx-independent manner.•MLN4924 also inhibits the synthesis of viral transcripts from episomal cccDNA.
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