scholarly journals Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Armando Arias ◽  
Lucy Thorne ◽  
Elsa Ghurburrun ◽  
Dalan Bailey ◽  
Ian Goodfellow
Keyword(s):  
Genetic Diversity ◽  
Virus Transmission ◽  
Viral Pathogenesis ◽  
High Fidelity ◽  
Wild Type ◽  
Replication Fidelity ◽  
Polymerase Fidelity ◽  
Natural Hosts ◽  
Insight Into

ABSTRACT Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains. Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.

scholarly journals Polymerase Fidelity Contributes to Foot-and-Mouth Disease Virus Pathogenicity and Transmissibility In Vivo

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Chen Li ◽  
Jiabao Shi ◽  
Haiwei Wang ◽  
Efraín E. Rivera-Serrano ◽  
Decheng Yang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Amino Acid ◽  
Foot And Mouth Disease ◽  
Single Amino Acid ◽  
Wild Type ◽  
High Genetic Diversity ◽  
Polymerase Fidelity ◽  
Mouth Disease Virus ◽  
Natural Hosts

ABSTRACT The low fidelity of foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase allows FMDV to exhibit high genetic diversity. Previously, we showed that the genetic diversity of FMDV plays an important role in virulence in suckling mice. Here, we mutated the amino acid residue Phe257, located in the finger domain of FMDV polymerase and conserved across FMDV serotypes, to a cysteine (F257C) to study the relationship between viral genetic diversity, virulence, and transmissibility in natural hosts. The single amino acid substitution in FMDV polymerase resulted in a high-fidelity virus variant, rF257C, with growth kinetics indistinguishable from those of wild-type (WT) virus in cell culture, but it displayed smaller plaques and impaired fitness in direct competition assays. Furthermore, we found that rF257C was attenuated in vivo in both suckling mice and pigs (one of its natural hosts). Importantly, contact exposure experiments showed that the rF257C virus exhibited reduced transmissibility compared to that of wild-type FMDV in the porcine model. This study provides evidence that FMDV genetic diversity is important for viral virulence and transmissibility in susceptible animals. Given that type O FMDV exhibits the highest genetic diversity among all seven serotypes of FMDV, we propose that the lower polymerase fidelity of the type O FMDV could contribute to its dominance worldwide. IMPORTANCE Among the seven serotypes of FMDV, serotype O FMDV have the broadest distribution worldwide, which could be due to their high virulence and transmissibility induced by high genetic diversity. In this paper, we generated a single amino acid substitution FMDV variant with a high-fidelity polymerase associated with viral fitness, virulence, and transmissibility in a natural host. The results highlight that maintenance of viral population diversity is essential for interhost viral spread. This study provides evidence that higher genetic diversity of type O FMDV could increase both virulence and transmissibility, thus leading to their dominance in the global epidemic.

scholarly journals Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

2016 ◽  
Author(s):  
Maureen M. Mundia ◽  
Alissa C. Magwood ◽  
Mark D. Baker
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Strand Exchange ◽  
Wild Type ◽  
Hybridoma Cell Lines ◽  
Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

scholarly journals Complete Genome Sequences of Measles Virus Genotype D8 Isolates from South Korea in 2016

2019 ◽  
Vol 8 (29) ◽  
Author(s):  
You-Jin Kim ◽  
Hae Ji Kang ◽  
Su-Jin Kim ◽  
Hye Min Lee ◽  
Sung Soon Kim
Keyword(s):  
Genetic Diversity ◽  
South Korea ◽  
Complete Genome ◽  
Measles Virus ◽  
Virus Transmission ◽  
Wild Type ◽  
Genome Sequences ◽  
Virus Genotype

The complete genome sequences of three wild-type measles viruses (genotype D8) isolated from patients in South Korea were determined. These are the first reported complete genome sequences of measles viruses obtained from South Korea, and the availability of these sequences will improve our understanding of measles virus transmission and genetic diversity.

scholarly journals Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

2014 ◽  
Vol 83 (1) ◽  
pp. 396-404 ◽  
Author(s):  
Jonathan F. Holt ◽  
Megan R. Kiedrowski ◽  
Kristi L. Frank ◽  
Jing Du ◽  
Changhui Guan ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Manduca Sexta ◽  
Wild Type ◽  
Genetic Determinants ◽  
Content Type ◽  
Polystyrene Plate ◽  
Insect Gut ◽  
Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

scholarly journals LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis

2021 ◽  
Vol 22 (18) ◽  
pp. 10117
Author(s):  
Hongjian Zhou ◽  
Shengjie Li ◽  
Shanshan Wu ◽  
Ping Jin ◽  
Fei Ma
Keyword(s):  
Immune Responses ◽  
Antimicrobial Peptide ◽  
Wild Type ◽  
Toll Signaling ◽  
Animal Survival ◽  
Non Coding Rna ◽  
Dynamic Expression ◽  
Long Non Coding Rna ◽  
Insight Into

Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. In this study, we firstly discover that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, our results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, our findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w1118) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, our study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity.

scholarly journals Using Barcoded Zika Virus to Assess Virus Population Structure in vitro and in Aedes aegypti Mosquitoes

2017 ◽  
Author(s):  
James Weger-Lucarelli ◽  
Selene M. Garcia ◽  
Claudia Rückert ◽  
Alex Byas ◽  
Shelby L. O’Connor ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Next Generation Sequencing ◽  
Aedes Aegypti ◽  
Zika Virus ◽  
Virus Transmission ◽  
Viral Diversity ◽  
Virus Population ◽  
Generation Sequencing

ABSTRACTArboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) replicate in both mammalian and insect hosts where they encounter a variety of distinct host defenses. To overcome these pressures, arboviruses exist as diverse populations of distinct genomes. However, transmission between hosts and replication within hosts can involve genetic bottlenecks, during which population size and viral diversity may be significantly reduced, potentially resulting in large fitness losses. Understanding the points at which bottlenecks exist during arbovirus transmission is critical to identifying targets for preventing transmission. To study these bottleneck effects, we constructed 4 “barcoded” ZIKV clones - 2 with an 8-base-pair degenerate insertion in the 3’ UTR and 2 with 8 or 9 degenerate synonymous changes in the coding sequence, theoretically containing thousands of variants each. We passaged these viruses 3 times each in 2 mammalian and 2 mosquito cell lines and characterized selection of the “barcode” populations using deep sequencing. Additionally, the viruses were used to feed three recently field-caught populations of Aedes aegypti mosquitoes to assess bottlenecks in a natural host. The barcoded viruses replicated well in multiple cell lines in vitro and in vivo in mosquitoes and could be characterized using next-generation sequencing. The stochastic nature of mosquito transmission was clearly shown by tracking individual barcodes in Ae. aegypti mosquitoes. Barcoded viruses provide an efficient method to examine bottlenecks during virus infection.AUTHOR SUMMARYIn general, mosquito-borne viruses like ZIKV must replicate in two very different host environments: an insect and a mammalian host. RNA viruses such as ZIKV must maintain genetic diversity in order to adapt to these changing conditions. During this transmission cycle, several barriers exist which can severely restrict viral genetic diversity, causing bottlenecks in the virus population. It is critical to understand these bottlenecks during virus transmission as this will provide important insights into the selective forces shaping arbovirus evolution within and between hots. Here, we employ a set of barcoded ZIKV constructs containing a degenerate stretch of nucleotides that can be tracked using next-generation sequencing. We found that the insertion site in the genome was an important determinant of the resulting diversity of the genetic barcode. We also found that bottlenecks varied between different mosquito populations and patterns of genetic diversity were distinct among individual mosquitoes within a single population, highlighting the randomness of virus dissemination in mosquitoes. Our study characterizes a new tool for tracking bottlenecks during virus transmission in vivo and highlights the importance of both viral and host factors on the maintenance of viral diversity.

scholarly journals Conserved Region 2.1 of Escherichia coli Heat Shock Transcription Factor σ32 Is Required for Modulating both Metabolic Stability and Transcriptional Activity

2004 ◽  
Vol 186 (22) ◽  
pp. 7474-7480 ◽  
Author(s):  
Mina Horikoshi ◽  
Takashi Yura ◽  
Sachie Tsuchimoto ◽  
Yoshihiro Fukumori ◽  
Masaaki Kanemori
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Heat Shock ◽  
Transcriptional Activity ◽  
Heat Shock Transcription Factor ◽  
Metabolic Stability ◽  
Wild Type ◽  
Shock Proteins ◽  
Insight Into

ABSTRACT Escherichia coli heat shock transcription factor σ32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of σ32. To gain further insight into the regulation of σ32 stability, we isolated σ32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial σ factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable σ32. Detailed analysis showed that some stable σ32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of σ32. The evidence suggests that σ32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.

scholarly journals Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tadasuke Naito ◽  
Kazumasa Shirai ◽  
Kotaro Mori ◽  
Hidetaka Muratsu ◽  
Hiroshi Ushirogawa ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Viruses ◽  
Viral Rna ◽  
Wild Type ◽  
Mutator Phenotype ◽  
Polymerase Fidelity

ABSTRACT In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivo. IMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.

scholarly journals PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Salman Mahmud Siddique ◽  
Koji Kubouchi ◽  
Yuka Shinmichi ◽  
Nana Sawada ◽  
Reiko Sugiura ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Kinase Activity ◽  
Extramedullary Hematopoiesis ◽  
Activation Loop ◽  
Wild Type ◽  
Phenotypic Differences ◽  
Dsdna Antibody ◽  
Independent Functions ◽  
Insight Into

Abstract Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.

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