scholarly journals Enterovirus 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 11 ◽  
Author(s):  
Zi-Wei Liu ◽  
Zhi-Chao Zhuang ◽  
Rui Chen ◽  
Xiao-Rui Wang ◽  
Hai-Lu Zhang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Virus Replication ◽  
Recombinant Virus ◽  
Enterovirus 71 ◽  
Nerve Cells ◽  
Full Length ◽  
Virulent Strains ◽  
Human Neurons ◽  
Virulence Attenuation ◽  
Full Length Gene

Background: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. Methods: A recombinant virus—SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01—was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. Results: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. Conclusions: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

scholarly journals Reverse Genetic Approaches for the Generation of Full Length and Subgenomic Replicon of EV71 Virus

2021 ◽  
Vol 12 ◽  
Author(s):  
Hang Yang ◽  
Xiaohui Zhao ◽  
Meng Xun ◽  
Chaofeng Ma ◽  
Hongliang Wang
Keyword(s):  
Viral Replication ◽  
Reporter Gene ◽  
Specific Treatment ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Full Length ◽  
Reverse Genetic ◽  
Structural Genes ◽  
Respiratory Complications ◽  
Subgenomic Replicon

Enterovirus 71 (EV71) is a neurotropic pathogen that causes hand, foot, and mouth disease (HFMD) and it has been consistently associated with severe neurological, cardiac, and respiratory complications. Yet there is no specific treatment for this virus and we still know little about the viral pathogenesis. In this study, we first generated an infectious cDNA clone of EV71 virus from a patient virus strain and made a full-length virus with a NanoLuc reporter gene through reverse genetic approaches. The reporter gene of this virus is genetically stable when passaging in cells and could be used for antiviral testing. In addition, we also made subgenomic replicons (SGRs) of EV71, which lacks part of the structural genes dispensable for viral replication and showed that SGR can be used for viral replication study. Overall, these reporter viral systems are useful tools for EV71 pathogenesis study and antiviral screening.

scholarly journals Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

2021 ◽  
Author(s):  
Makoto Ujike∗ ◽  
Yuka Etoh ◽  
Naoya Urushiyama ◽  
Fumihiro Taguchi ◽  
Hideki Asanuma ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cdna Clone ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Full Length ◽  
Egfp Expression ◽  
Egfp Gene ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Gene Retention

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses

2013 ◽  
Vol 194 (1-2) ◽  
pp. 82-88
Author(s):  
Britta Lassmann ◽  
Vaithilingaraja Arumugaswami ◽  
Kara W. Chew ◽  
Martha J. Lewis
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Full Length ◽  
Replication Capacity ◽  
New System

Enterovirus 71 induces integrin β1/EGFR-Rac1-dependent oxidative stress in SK-N-SH cells: Role of HO-1/CO in viral replication

2011 ◽  
Vol 226 (12) ◽  
pp. 3316-3329 ◽  
Author(s):  
Wei-Hsuan Tung ◽  
Hsi-Lung Hsieh ◽  
I-Ta Lee ◽  
Chuen-Mao Yang
Keyword(s):  
Oxidative Stress ◽  
Viral Replication ◽  
Enterovirus 71 ◽  
Integrin Β1

scholarly journals A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Niken M. Mahaweni ◽  
Timo I. Olieslagers ◽  
Ivan Olivares Rivas ◽  
Stefan J. J. Molenbroeck ◽  
Mathijs Groeneweg ◽  
...  
Keyword(s):  
Gene Sequencing ◽  
Full Length ◽  
Comprehensive Overview ◽  
Gene Variability ◽  
Full Length Gene

scholarly journals Network-Guided Discovery of Influenza Virus Replication Host Factors

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Emily E. Ackerman ◽  
Eiryo Kawakami ◽  
Manami Katoh ◽  
Tokiko Watanabe ◽  
Shinji Watanabe ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Virus Replication ◽  
Drug Targets ◽  
Antiviral Drug ◽  
Viral Proteins ◽  
Host Factors ◽  
Ppi Network ◽  
Host Proteins ◽  
Influenza Virus Replication

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

scholarly journals Murine Norovirus Replication Induces G0/G1Cell Cycle Arrest in Asynchronously Growing Cells

2015 ◽  
Vol 89 (11) ◽  
pp. 6057-6066 ◽  
Author(s):  
Colin Davies ◽  
Chris M. Brown ◽  
Dana Westphal ◽  
Joanna M. Ward ◽  
Vernon K. Ward
Keyword(s):  
Cell Cycle ◽  
Viral Replication ◽  
Host Cell ◽  
Virus Replication ◽  
Cell Cycle Progression ◽  
Cycle Phase ◽  
Murine Norovirus ◽  
Cycle Progression ◽  
Infected Cells ◽  
Favorable Conditions

ABSTRACTMany viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1phase and an accumulation of cells in the G0/G1phase. The accumulation in G0/G1phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1phase may be representative of other members of theCaliciviridae.IMPORTANCENoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1phase. Furthermore, we show that MNV replication is enhanced in the G1phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1phase for RNA virus replication.

scholarly journals Characterization of Full-Length Enterovirus 71 Strains from Severe and Mild Disease Patients in Northeastern China

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e32405 ◽  
Author(s):  
Xiaomei Wang ◽  
Chunfeng Zhu ◽  
Wanguo Bao ◽  
Ke Zhao ◽  
Junqi Niu ◽  
...  
Keyword(s):  
Enterovirus 71 ◽  
Northeastern China ◽  
Full Length ◽  
Mild Disease

Association of the IVR Gene with Virus Localization and Resistance

1995 ◽  
Author(s):  
Gad Loebenstein ◽  
William Dawson ◽  
Abed Gera
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Plant Viruses ◽  
Full Length ◽  
N Gene ◽  
Complete Suppression ◽  
Coat Proteins ◽  
Nicotiana Glutinosa ◽  
Nicotiana Debneyi ◽  
Full Length Gene

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.

EV71 2A Protease inhibits P-body formation to promote viral RNA synthesis

2021 ◽  
Author(s):  
Shanshan Fan ◽  
Zihang Xu ◽  
Pengfei Liu ◽  
Yali Qin ◽  
Mingzhou Chen
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Enterovirus 71 ◽  
Viral Rna ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Body Components ◽  
2A Protease ◽  
Enterovirus 71 Infection

Several viruses were proved to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proved by the result that infection of EV71-2A C110S , the 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we showed DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and capturing the nascent RNA assay. Altogether, our data firstly demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulating gene expression and mRNA degradation. P-bodies are the structure that viruses to manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection and its activity was essential. When the assembly of P-bodies was blocked by 2A, DDX6 and 4E-T which were required for P-body formation bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

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