Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B6 precursor, and indoles. Growth restriction of C. parvum by indoles does not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impairs host mitochondrial function and reduces total cellular ATP, as well as directly reducing the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole-producing bacteria, delays life cycle progression of the parasite in vitro and reduces the severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites impair mitochondrial function and contribute to colonization resistance to Cryptosporidium infection.

Selective Polyprotein Processing Determines Norovirus Sensitivity to Trim7.

Noroviruses are a leading cause of gastroenteritis worldwide, yet the molecular mechanisms of how host antiviral factors restrict norovirus infection are poorly understood. Here, we present a CRISPR activation screen that identifies mouse genes which inhibit murine norovirus (MNV) replication. Detailed analysis of the major hit Trim7 demonstrates a potent inhibition of the early stages of MNV replication. Leveraging in vitro evolution, we identified MNV mutants that escape Trim7 restriction by altering the cleavage of the viral NS6-7 polyprotein precursor. NS6, but not the NS6-7 precursor, directly binds the substrate-binding domain of Trim7. Surprisingly, the selective polyprotein processing that enables Trim7 evasion inflicts a significant evolutionary burden, as viruses with decreased NS6-7 cleavage are strongly attenuated in viral replication and pathogenesis. Our data provide an unappreciated mechanism of viral evasion of cellular antiviral factors through selective polyprotein processing and highlight the evolutionary tradeoffs in acquiring resistance to host restriction factors. IMPORTANCE To maximize a limited genetic capacity, viruses encode polyproteins that can be subsequently separated into individual components by viral proteases. While classically viewed as a means of economy, recent findings have indicated that polyprotein processing can spatially and temporally coordinate the distinct phases of the viral life cycle. Here, we present a function for alternative polyprotein processing centered on immune defense. We discovered that selective polyprotein processing of the murine norovirus polyprotein shields MNV from restriction by the host antiviral protein Trim7. Trim7 can bind the viral protein NS6 but not the viral precursor protein NS6-7. Our findings provide insight into the evolutionary pressures that define patterns of viral polyprotein processing and uncover a trade-off between viral replication and immune evasion.

Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

A modified bacterial one-hybrid system yields improved quantitative models of transcription factor specificity.

We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis--GRaMS (Growth Rate Modeling of Specificity)--that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well-characterized C(2)H(2) zinc-finger TF on both a 28 bp randomized library for the standard B1H method and on 6 bp randomized library for the CV-B1H method for which 45 different experimental conditions were tested: five time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media.

Caenorhabditis elegans unc-82 encodes a serine/threonine kinase important for myosin filament organization in muscle during growth.

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.

Identification of muscle-specific regulatory modules in Caenorhabditis elegans.

Transcriptional regulation is the major regulatory mechanism that controls the spatial and temporal expression of genes during development. This is carried out by transcription factors (TFs), which recognize and bind to their cognate binding sites. Recent studies suggest a modular organization of TF-binding sites, in which clusters of transcription-factor binding sites cooperate in the regulation of downstream gene expression. In this study, we report our computational identification and experimental verification of muscle-specific cis-regulatory modules in Caenorhabditis elegans. We first identified a set of motifs that are correlated with muscle-specific gene expression. We then predicted muscle-specific regulatory modules based on clusters of those motifs with characteristics similar to a collection of well-studied modules in other species. The method correctly identifies 88% of the experimentally characterized modules with a positive predictive value of at least 65%. The prediction accuracy of muscle-specific expression on an independent test set is highly significant (P<0.0001). We performed in vivo experimental tests of 12 predicted modules, and 10 of those drive muscle-specific gene expression. These results suggest that our method is highly accurate in identifying functional sequences important for muscle-specific gene expression and is a valuable tool for guiding experimental designs.

Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development.

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in Caenorhabditis elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. We find that two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naïve blastomeres to muscle when overproduced ectopically in the embryo. In addition, we have used genetic null mutants to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type.

Novel transcription regulatory elements in Caenorhabditis elegans muscle genes.

We report the identification of three new transcription regulatory elements that are associated with muscle gene expression in the nematode Caenorhabditis elegans. Starting from a subset of well-characterized nematode muscle genes, we identified conserved DNA motifs in the promoter regions using computational DNA pattern-recognition algorithms. These were considered to be putative muscle transcription regulatory motifs. Using the green-fluorescent protein (GFP) as a reporter, experiments were done to determine the biological activity of these motifs in driving muscle gene expression. Prediction accuracy of muscle expression based on the presence of these three motifs was encouraging; nine of 10 previously uncharacterized genes that were predicted to have muscle expression were shown to be expressed either specifically or selectively in the muscle tissues, whereas only one of the nine that scored low for these motifs expressed in muscle. Knockouts of putative regulatory elements in the promoter of the mlc-2 and unc-89 genes show that they significantly contribute to muscle expression and act in a synergistic manner. We find that these DNA motifs are also present in the muscle promoters of C. briggsae, indicating that they are functionally conserved in the nematodes.

mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament.

Locomotion in Caenorhabditis elegans requires force transmission through a network of proteins linking the skeletal muscle, via an intervening basal lamina and epidermis (hypodermis), to the cuticle. Mutations in mua-6 result in hypodermal rupture, muscle detachment from the bodywall, and progressive paralysis. It is shown that mua-6 encodes the cytoplasmic intermediate filament (cIF) A2 protein and that a MUA-6/IFA-2::GFP fusion protein that rescues the presumptive mua-6 null allele localizes to hypodermal hemidesmosomes. This result is consistent with what is known about the function of cIFs in vertebrates. Although MUA-6/IFA-2 is expressed embryonically, and plays an essential postembryonic role in tissue integrity, it is not required for embryonic development of muscle-cuticle linkages nor for the localization of other cIFs or hemidesmosome-associated proteins in the embryo. Finally, the molecular lesion in the mua-6(rh85) allele suggests that the head domain of the MUA-6/IFA-2 is dispensable for its function.