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Unlabelled: Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex., Importance: RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Motivation: The study of RNA virus populations is a challenging task. Each population of RNA virus is composed of a collection of different, yet related genomes often referred to as mutant spectra or quasispecies. Virologists using deep sequencing technologies face major obstacles when studying virus population dynamics, both experimentally and in natural settings due to the relatively high error rates of these technologies and the lack of high performance pipelines. In order to overcome these hurdles we developed a computational pipeline, termed ViVan (Viral Variance Analysis). ViVan is a complete pipeline facilitating the identification, characterization and comparison of sequence variance in deep sequenced virus populations., Results: Applying ViVan on deep sequenced data obtained from samples that were previously characterized by more classical approaches, we uncovered novel and potentially crucial aspects of virus populations. With our experimental work, we illustrate how ViVan can be used for studies ranging from the more practical, detection of resistant mutations and effects of antiviral treatments, to the more theoretical temporal characterization of the population in evolutionary studies., Availability and Implementation: Freely available on the web at http://www.vivanbioinfo.org, Contact: : nshomron@post.tau.ac.il, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press.)

Based on structural data of the RNA-dependent RNA polymerase, rational targeting of key residues, and screens for Coxsackievirus B3 fidelity variants, we isolated nine polymerase variants with mutator phenotypes, which allowed us to probe the effects of lowering fidelity on virus replication, mutability, and in vivo fitness. These mutator strains generate higher mutation frequencies than WT virus and are more sensitive to mutagenic treatments, and their purified polymerases present lower-fidelity profiles in an in vitro incorporation assay. Whereas these strains replicate with WT-like kinetics in tissue culture, in vivo infections reveal a strong correlation between mutation frequency and fitness. Variants with the highest mutation frequencies are less fit in vivo and fail to productively infect important target organs, such as the heart or pancreas. Furthermore, whereas WT virus is readily detectable in target organs 30 d after infection, some variants fail to successfully establish persistent infections. Our results show that, although mutator strains are sufficiently fit when grown in large population size, their fitness is greatly impacted when subjected to severe bottlenecking, which would occur during in vivo infection. The data indicate that, although RNA viruses have extreme mutation frequencies to maximize adaptability, nature has fine-tuned replication fidelity. Our work forges ground in showing that the mutability of RNA viruses does have an upper limit, where larger than natural genetic diversity is deleterious to virus survival.

The error rate of RNA-dependent RNA polymerases (RdRp) affects the mutation frequency in a population of viral RNAs. Using chikungunya virus (CHIKV), we describe a unique arbovirus fidelity variant with a single C483Y amino acid change in the nsP4 RdRp that increases replication fidelity and generates populations with reduced genetic diversity. In mosquitoes, high fidelity CHIKV presents lower infection and dissemination titers than wild type. In newborn mice, high fidelity CHIKV produces truncated viremias and lower organ titers. These results indicate that increased replication fidelity and reduced genetic diversity negatively impact arbovirus fitness in invertebrate and vertebrate hosts.

Most cases of juvenile-onset diabetes (JOD) are diagnosed as type 1 diabetes (T1D), for which genetic studies conducted in outbred Caucasian populations support the concept of multifactorial inheritance. However, this view may be partly challenged in particular population settings. In view of the suggestive evidence for a high prevalence of Wolfram syndrome (WFS) in Lebanon, the phenotypic variability associated with WFS1 mutations, and the high consanguinity rate in Lebanon, we aimed to evaluate the contribution of WFS1 mutations as monogenic determinants to JOD in Lebanon. We performed a family-based genetic study, with linkage analysis followed by systematic mutation screening of WFS1 exons in all JOD probands. The study population consisted of an unbiased recruitment of all juvenile-onset insulin-dependent diabetic patients from a specialized diabetes pediatric clinic in Beirut, Lebanon. Homozygous or compound heterozygous WFS1 mutations were found in 22 of the 399 JOD probands (5.5%), resulting in WFS (17 probands) or in non-syndromic non-autoimmune diabetes mellitus (DM, five probands). These accounted for 12.1% (21/174) of probands in consanguineous families, compared with 0.4% (1/225) in non-consanguineous families. Of the 38 patients identified with homozygous or compound heterozygous WFS1 mutations, 11 (29%) had non-syndromic DM, all of whom carried a particular WFS1 mutation, WFS1(LIB), encoding a protein with an extended C-terminal domain. This mutation resulted in a delayed onset or absence of extrapancreatic features. These results underscore the major impact of population-specific factors, such as population-specific mutations and founder effects, and family structure in the genetic determinism of JOD.

We recently described a new neonatal diabetes syndrome associated with congenital hypothyroidism, congenital glaucoma, hepatic fibrosis and polycystic kidneys. Here, we show that this syndrome results from mutations in GLIS3, encoding GLI similar 3, a recently identified transcription factor. In the original family, we identified a frameshift mutation predicted to result in a truncated protein. In two other families with an incomplete syndrome, we found that affected individuals harbor deletions affecting the 11 or 12 5'-most exons of the gene. The absence of a major transcript in the pancreas and thyroid (deletions from both families) and an eye-specific transcript (deletion from one family), together with residual expression of some GLIS3 transcripts, seems to explain the incomplete clinical manifestations in these individuals. GLIS3 is expressed in the pancreas from early developmental stages, with greater expression in beta cells than in other pancreatic tissues. These results demonstrate a major role for GLIS3 in the development of pancreatic beta cells and the thyroid, eye, liver and kidney.