scholarly journals Hsp70 Negatively Controls Rotavirus Protein Bioavailability in Caco-2 Cells Infected by the Rotavirus RF Strain

2006 ◽  
Vol 81 (3) ◽  
pp. 1297-1304 ◽  
Author(s):  
Alexis H. Broquet ◽  
Christelle Lenoir ◽  
Agnès Gardet ◽  
Catherine Sapin ◽  
Serge Chwetzoff ◽  
...  
Keyword(s):  
Viral Infection ◽  
Proteasome Inhibitors ◽  
Virus Production ◽  
Viral Proteins ◽  
Host Cells ◽  
Progeny Virus ◽  
Virus Protein ◽  
Interfering Rna ◽  
Hsp70 Induction

ABSTRACT Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.

scholarly journals Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

2008 ◽  
Vol 89 (8) ◽  
pp. 1873-1880 ◽  
Author(s):  
Qian Yu ◽  
Tiehao Lin ◽  
Guozhong Feng ◽  
Kai Yang ◽  
Yi Pang
Keyword(s):  
Spodoptera Litura ◽  
Virus Production ◽  
Host Cells ◽  
Homology Search ◽  
Pcr Analysis ◽  
Viability Analysis ◽  
Infected Cells ◽  
Budded Virus ◽  
Almost All

A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.

scholarly journals Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Nonstructural Protein ◽  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

ABSTRACT Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions. IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1881-1888 ◽  
Author(s):  
L. M. Kasman ◽  
L. E. Volkman
Keyword(s):  
Helicoverpa Zea ◽  
Virus Production ◽  
Host Cells ◽  
Actin Binding ◽  
Anticarsia Gemmatalis ◽  
Progeny Virus ◽  
Filamentous Actin ◽  
Phylogenetic Groups ◽  
Dna Viruses ◽  
Orgyia Pseudotsugata

Autographa californica M nucleopolyhedrovirus (AcMNPV) is the prototypical member of the Nucleopolyhedrosis genus of the Baculoviridae, a family of large, double-stranded DNA viruses that are highly diverse. Nucleocapsid morphogenesis of AcMNPV and others in the Nucleopolyhedrovirus genus takes place within the nuclei of infected host cells. Previously, we showed that filamentous actin (F-actin) is essential for this process to occur in AcMNPV-infected cells, an unprecedented finding for a DNA virus that replicates within the nucleus. Because of the fundamental importance of this requirement to our understanding of virus–host interactions, and because of the diversity of viruses included within the Nucleopolyhedrovirus genus, we were compelled to determine whether the replication of other nucleopolyhedroviruses was also F-actin dependent. We report here that progeny virus production of six other lepidopteran nucleopolyhedroviruses, representing both phylogenetic groups I and II within the genus, is also F-actin dependent. The six viruses studied (Spodoptera frugiperda MNPV, Bombyx mori NPV, Orgyia pseudotsugata MNPV, Lymantria dispar MNPV, Anticarsia gemmatalis MNPV and Helicoverpa zea SNPV) were unable to produce progeny in the presence of either cytochalasin D or latrunculin A, two actin-binding agents that interfere with F-actin-dependent processes but differ in their modes of action. F-actin-dependent progeny morphogenesis, therefore, appears to be a characteristic common among viruses in this genus that have lepidopteran hosts.

scholarly journals Clarithromycin Inhibits Progeny Virus Production from Human Influenza Virus-Infected Host Cells

2008 ◽  
Vol 31 (2) ◽  
pp. 217-222 ◽  
Author(s):  
Daisei Miyamoto ◽  
Sayaka Hasegawa ◽  
Nongluk Sriwilaijaroen ◽  
Sangchai Yingsakmongkon ◽  
Hiroaki Hiramatsu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Production ◽  
Host Cells ◽  
Infected Host ◽  
Progeny Virus ◽  
Human Influenza ◽  
Human Influenza Virus ◽  
Progeny Virus Production

scholarly journals Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by InhibitingBAD-Mediatedcaspase-3-Dependent Apoptosis

2015 ◽  
Vol 90 (3) ◽  
pp. 1359-1368 ◽  
Author(s):  
Hyoji Kim ◽  
Hoyun Choi ◽  
Suk Kyeong Lee
Keyword(s):  
Epstein Barr Virus ◽  
Caspase 3 ◽  
Immune Surveillance ◽  
Virus Production ◽  
Lytic Cycle ◽  
Host Cells ◽  
Immediate Early ◽  
Progeny Virus ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and thein vivotriggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genesBRLF1andBZLF1as well asBcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels ofBRLF1andBZLF1were suppressed in cells followingBADknockdown and increased afterBADoverexpression. Progeny virus production was also downregulated by specific knockdown ofBAD. Our results demonstrated thatcaspase-3-dependent apoptosis is a prerequisite forBAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibitingBAD-mediatedcaspase-3-dependent apoptosis, which would trigger immediate early gene expression.IMPORTANCEEBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressingBAD-inducedcaspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

scholarly journals Identification of TRAIL as an Interferon Regulatory Factor 3 Transcriptional Target

2005 ◽  
Vol 79 (14) ◽  
pp. 9320-9324 ◽  
Author(s):  
Jessica R. Kirshner ◽  
Alla Y. Karpova ◽  
Maren Kops ◽  
Peter M. Howley
Keyword(s):  
Viral Infection ◽  
Virus Production ◽  
Interferon Regulatory Factor ◽  
Progeny Virus ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Apoptosis Pathway ◽  
Infected Cells ◽  
Transcriptional Induction ◽  
Transcriptional Target

ABSTRACT Interferon production and apoptosis in virus-infected cells are necessary to prevent progeny virus production and to eliminate infected cells. Paramyxovirus infection induces apoptosis through interferon regulatory factor 3 (IRF-3), but the exact mechanism of how IRF-3 functions is unknown. We show that IRF-3 is involved in the transcriptional induction of TRAIL, a key player in the apoptosis pathway. IRF-3 upregulates TRAIL transcription following viral infection and binds an interferon-stimulated response element in the TRAIL promoter. The mRNA for TRAIL and its receptor, DR5, are induced following viral infection. These studies identify TRAIL as a novel IRF-3 transcriptional target.

Cellular and molecular biology of HCV infection and hepatitis

2009 ◽  
Vol 117 (2) ◽  
pp. 49-65 ◽  
Author(s):  
Hengli Tang ◽  
Henry Grisé
Keyword(s):  
Hepatitis C ◽  
Rna Replication ◽  
Viral Proteins ◽  
The United States ◽  
Hcv Infection ◽  
Host Cells ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Positive Strand Rna

HCV (hepatitis C virus) infects nearly 3% of the population worldwide and has emerged as a major causative agent of liver disease, resulting in acute and chronic infections that can lead to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatitis C represents the leading cause of liver transplantation in the United States and Europe. A positive-strand RNA virus of the Flaviviridae family, HCV contains a single-stranded RNA genome of approx. 9600 nucleotides. The genome RNA serves as both mRNA for translation of viral proteins and the template for RNA replication. Cis-acting RNA elements within the genome regulate RNA replication by forming secondary structures that interact with each other and trans-acting factors. Although structural proteins are clearly dispensable for RNA replication, recent evidence points to an important role of several non-structural proteins in particle assembly and release, turning their designation on its head. HCV enters host cells through receptor-mediated endocytosis, and the process requires the co-ordination of multiple cellular receptors and co-receptors. RNA replication takes place at specialized intracellular membrane structures called ‘membranous webs’ or ‘membrane-associated foci’, whereas viral assembly probably occurs on lipid droplets and endoplasmic reticulum. Liver inflammation plays a central role in the liver damage seen in hepatitis C, but many HCV proteins also directly contribute to HCV pathogenesis. In the present review, the molecular and cellular aspects of the HCV life cycle and the role of viral proteins in pathological liver conditions caused by HCV infection are described.

scholarly journals Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

2017 ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Structural Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

AbstractWithin infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, μNS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.ImportanceMRV has historically been used as a model to study the double-stranded RNA (dsRNA)Reoviridaefamily, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein μNS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

scholarly journals Comprehensive analysis of the host-virus interactome of SARS-CoV-2

2021 ◽  
Author(s):  
Zhen Chen ◽  
Chao Wang ◽  
Xu Feng ◽  
Litong Nie ◽  
Mengfan Tang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Viral Proteins ◽  
Host Cells ◽  
Virus Protein ◽  
N Protein ◽  
Protein Protein Interaction ◽  
Human Coronaviruses

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

scholarly journals Role of Endoplasmic Reticulum-Associated Proteins in Flavivirus Replication and Assembly Complexes

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 148 ◽  
Author(s):  
Hussin A. Rothan ◽  
Mukesh Kumar
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Proteins ◽  
Host Cells ◽  
Host Proteins ◽  
Multiprotein Complexes ◽  
Genome Wide ◽  
Associated Proteins ◽  
Cytoplasmic Side ◽  
Er Membrane

Flavivirus replication in host cells requires the formation of replication and assembly complexes on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. These complexes consist of an ER membrane, viral proteins, and host proteins. Genome-wide investigations have identified a number of ER multiprotein complexes as vital factors for flavivirus replication. The detailed mechanisms of the role of ER complexes in flavivirus replication are still largely elusive. This review highlights the fact that the ER multiprotein complexes are crucial for the formation of flavivirus replication and assembly complexes, and the ER complexes could be considered as a target for developing successful broad-spectrum anti-flavivirus drugs.

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