scholarly journals Mutational Disruption ofcis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions

2015 ◽  
Vol 89 (23) ◽  
pp. 11761-11772 ◽  
Author(s):  
S. Smithee ◽  
S. Tracy ◽  
N. M. Chapman
Keyword(s):  
Rna Structure ◽  
De Novo ◽  
Virus Genome ◽  
Viral Rna ◽  
Sequence Information ◽  
Virus Population ◽  
Wild Type ◽  
Coding Region ◽  
Free System ◽  
Genomic Deletions

ABSTRACTFollowing natural human or experimental murine infections and in cell culture, coxsackievirus B (CVB) RNA can persist for weeks in the absence of a cytopathic effect, yet viral RNA remains detectable. Our earlier studies demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the genomic 5′ terminus which partially degraded the cloverleaf (or domain I), an RNA structure required for efficient viral replication. Acis-acting replication element (CRE) in the 2C protein-coding region [CRE(2C)] templates the addition of two uridine residues to the virus genome-encoded RNA replication primer VPg prior to positive-strand synthesis. Because our previous work also demonstrated that the genomes of CVB with a 5′-terminal deletion (CVB-TD) have VPg covalently linked, even though they rarely terminate in the canonical UU donated by CRE(2C)-mediated uridylylation of VPg, we hypothesized that a functional (uridylylating) CRE(2C) would be unnecessary for CVB-TD replication. Using the same 16 mutations in the CVB3 CRE(2C) structure that were considered lethal for this virus by others, we demonstrate here both in infected cell cultures and in mice that wild-type (wt) and CVB3-TD strains carrying these mutations with a nonuridylylating CRE(2C) are viable. While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to those observed in a CVB3-TD strain, mutation of the CRE(2C) function in a CVB3-TD strain does not further decrease replication. Finally, we show that replication of the parental CVB3 strain containing the mutated CRE(2C) drives thede novogeneration of genomic deletions at the 5′ terminus.IMPORTANCEIn this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), the replication rate suffers significantly. Further, deletions at the 5′ terminus of the genome are generated in this virus population, with this virus population supplanting the wild-type population. This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in the loss of sequence information from the 5′ terminus. These findings have significance when considering the replication of human enteroviruses, and we believe that these data are unattainable in a cell-free system due to the poor replication of these CRE-deficient viruses.

scholarly journals Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 749 ◽  
Author(s):  
Melanie Hiltbrunner ◽  
Gerald Heckel
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Phylogenetic Analyses ◽  
Common Vole ◽  
Natural Host ◽  
Sequence Information ◽  
Coding Region ◽  
Sequence Capture ◽  
Genome Wide ◽  
The Impact

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

scholarly journals A subpopulation of RNA 1 of Cucumber mosaic virus contains 3′ termini originating from RNAs 2 or 3

2001 ◽  
Vol 82 (4) ◽  
pp. 941-945 ◽  
Author(s):  
Tomas Canto ◽  
Seung Kook Choi ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Viral Rna ◽  
Template Switching ◽  
Wild Type ◽  
Coding Region ◽  
Tobacco Plants ◽  
Viral Rnas ◽  
Virus Cdna ◽  
Cdna Fragments

Tobacco plants transgenic for RNA 1 of Cucumber mosaic virus and inoculated with transcript of RNAs 2 and 3 regenerated viral RNA 1 from the transgenic mRNA, and the plants became systemically infected by the reconstituted virus. cDNA fragments corresponding to the 3′ non-coding region (NCR) of viral RNA 1 were amplified, cloned and sequenced. In some clones the termini of the 3′ NCR corresponded to those of viral RNAs 2 or 3. This suggested that in some cases RNA 1 may have been regenerated during replication by a template switching mechanism between the inoculated transcript RNAs and the mRNA. However, encapsidated, recombinant RNA 1 with the 3′ NCR ends originating from RNAs 2 or 3 also was found in virus samples that had been passaged exclusively through non-transgenic plants. Thus, these chimeras occur naturally due to recombination between wild-type viral RNAs, and they are found encapsidated in low, but detectable amounts.

scholarly journals Sendai virus genome synthesis and assembly are coupled: a possible mechanism to promote viral RNA polymerase processivity

2001 ◽  
Vol 82 (12) ◽  
pp. 2895-2903 ◽  
Author(s):  
Oliver Gubbay ◽  
Joseph Curran ◽  
Daniel Kolakofsky
Keyword(s):  
Sendai Virus ◽  
Virus Genome ◽  
Rna Polymerases ◽  
Viral Rna ◽  
Reaction Conditions ◽  
Free System ◽  
N Protein ◽  
Cell Extracts ◽  
Virus Rna ◽  
L Proteins

A cell-free system for studying Sendai virus RNA synthesis was reconstituted from N protein:RNA templates and transfected cell extracts in which the viral N, P and L proteins were expressed. Both transcription (mRNA synthesis) and replication (genome and antigenome synthesis) took place concurrently in these reactions. Viral RNA polymerases engaged in replication (replicases) were found to elongate their chains at a constant speed along the genome (1·7 nt/s), in a highly processive manner. In contrast, viral RNA polymerases engaged in transcription (transcriptases), although capable of synthesizing RNA at a comparable speed to replicases, were poorly processive. In this system, therefore, transcriptases require special reaction conditions to promote processivity that are not required by replicases. In addition, during replication, incomplete nascent genome chains were shown to be assembled with N protein, providing direct evidence that the synthesis and assembly of genomes are concurrent events. The strong processivity of replicases, independent of the reaction conditions, may thus be due to the coupling of genome synthesis and assembly. A model is proposed to explain how pausing of viral polymerase on the template is restricted when assembly and synthesis of the nascent chain are coupled.

Growth properties of afolAnull mutant ofEscherichia coliK12

1999 ◽  
Vol 45 (3) ◽  
pp. 191-200 ◽  
Author(s):  
Muriel B Herrington ◽  
Neema T Chirwa
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Coding Region ◽  
Growth Properties ◽  
One Carbon Metabolism

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine- (thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions. Key words: dihydrofolate reductase, Escherichia coli, biosynthesis, folates, one-carbon metabolism.

scholarly journals Evolutionary selection on synonymous codons in RNA G-quadruplex structural region

2021 ◽  
Author(s):  
Yuming Xu ◽  
Ting Qi ◽  
Zuhong Lu ◽  
Tong Zhou ◽  
Wanjun Gu
Keyword(s):  
Rna Structure ◽  
Nucleotide Composition ◽  
Sequence Information ◽  
Coding Region ◽  
Protein Coding ◽  
Translational Initiation ◽  
Evolutionary Selection ◽  
Synonymous Codons ◽  
G Quadruplex ◽  
Structural Region

ABSTRACTIn addition to the amino acid sequence information, synonymous codons can encode multiple regulatory and structural signals in protein coding region. In this study, we investigated how synonymous codons have been adapted to the formation of RNA G-quadruplex (rG4) structure. We found a universal selective pressure acting on synonymous codons to facilitate rG4 formation in five eukaryotic organisms. While G-rich codons are preferred in rG4 structural region, C-rich codons are selectively unpreferred for rG4 structures. Gene’s codon usage bias, nucleotide composition and evolutionary rate can account for the selective variations on synonymous codons among rG4 structures within a species. Moreover, rG4 structures in translational initiation region showed significantly higher selective pressures than those in translational elongation region. These results bring us another dimension of evolutionary selection on synonymous codons for proper RNA structure and function.

scholarly journals Reduction of the Rate of Poliovirus Protein Synthesis through Large-Scale Codon Deoptimization Causes Attenuation of Viral Virulence by Lowering Specific Infectivity

2006 ◽  
Vol 80 (19) ◽  
pp. 9687-9696 ◽  
Author(s):  
Steffen Mueller ◽  
Dimitris Papamichail ◽  
J. Robert Coleman ◽  
Steven Skiena ◽  
Eckard Wimmer
Keyword(s):  
Large Scale ◽  
De Novo ◽  
Synonymous Codon ◽  
Direct Analysis ◽  
Synonymous Codon Usage ◽  
Wild Type ◽  
Coding Region ◽  
Virus Particles ◽  
Viral Virulence ◽  
Wide Range

ABSTRACT Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.

scholarly journals Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 6103-6118 ◽  
Author(s):  
Peter Friebe ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Rna Structure ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Coding Region ◽  
Stem Loop ◽  
Complementary Sequences ◽  
Translation Start ◽  
Rna Elements

ABSTRACT Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.

Occurrence and regression of BK polyomavirus associated carcinoma: a clinical and next-generation sequencing study

2018 ◽  
Vol 132 (16) ◽  
pp. 1753-1763 ◽  
Author(s):  
Fangxiang Fu ◽  
Wenfeng Deng ◽  
Siyuan Yu ◽  
Yanna Liu ◽  
Lixin Yu ◽  
...  
Keyword(s):  
De Novo ◽  
Virus Genome ◽  
T Antigen ◽  
The Body ◽  
Coding Region ◽  
Metastatic Lesions ◽  
Non Coding Rna ◽  
Bk Polyomavirus ◽  
Male Donor ◽  
Immunocompetent Adults

Low-level BK polyomavirus (BKPyV) shedding is seen in at least 10% of seropositive immunocompetent adults. Moreover, BKPyV infection is highly prevalent amongst immunocompromised populations, yet little is known on its relationship with malignancy. We studied a female patient with BKPyV-associated and donor-derived de novo high-grade sarcomatoid urothelial carcinoma developed 8 years after kidney transplantation from a male donor. Through whole-genome sequencing, we discovered integration of genotype IV BKPyV genome into the non-coding RNA (ncRNA) intronic region of human chromosome 18. The two breakpoints in the virus genome were located at the non-coding control region (NCCR) and large T antigen (TAg) coding region, respectively. Nevertheless, the TAg was overexpressed. We, therefore, inferred that the BKPyV was clonally integrated into the human genome in the form of concatemers, facilitating the expression of the TAg. The patient presented with multiorgan metastases, which were reduced in size and number throughout the body after removal of the graft and cessation of immunosuppressants. The few remaining lesions located in the liver were identified, through biopsy to be necrotic tumor tissue with TAg detected; additionally, genomic sequencing of the liver mass found Y chromosome. In conclusion, we propose that integration of the BKPyV genome is closely related to oncogenesis in this patient; while oncogenesis occurred when host immunity was impaired, recovery of the patient’s native immunity effectively curbed viral replication and eliminated the metastatic lesions.

scholarly journals Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

2021 ◽  
Author(s):  
Renato Salazar ◽  
Barbara Arbeithuber ◽  
Maja Ivankovic ◽  
Monika Heinzl ◽  
Sofia Moura ◽  
...  
Keyword(s):  
De Novo ◽  
Age Groups ◽  
Low Frequency ◽  
Congenital Disorders ◽  
Sequence Information ◽  
De Novo Mutations ◽  
Coding Region ◽  
Male Germline ◽  
Significant Difference ◽  
Duplex Sequencing

AbstractDe novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

scholarly journals Nucleotide Sequence Requirements at the 5′ End of the Influenza A Virus M RNA Segment for Efficient Virus Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3384-3388 ◽  
Author(s):  
Makoto Ozawa ◽  
Junko Maeda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
Shinji Watanabe ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Viral Rna ◽  
Coding Region ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Sequence Requirements

ABSTRACT The mechanism by which the influenza A virus genome is packaged into virions is not fully understood. The coding and noncoding regions necessary for packaging of the viral RNA segments, except for the M segment, have been identified. Here, we delineate the M segment regions by incorporating a reporter viral RNA into virions and by generating viruses possessing mutations in the regions. We found that, like the other segments, the M segment coding regions are essential for virion incorporation and that the nucleotide length rather than the nucleotide sequence of the 5′ end of the coding region is important.

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