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 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.

scholarly journals Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sithumini M. W. Lokupathirage ◽  
Yoshimi Tsuda ◽  
Kodai Ikegame ◽  
Kisho Noda ◽  
Devinda S. Muthusinghe ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Subcellular Localization ◽  
Critical Role ◽  
Viral Proteins ◽  
Viral Rna ◽  
Host Cells ◽  
N Protein ◽  
L Protein ◽  
Translational Activity ◽  
Antiviral Targets

AbstractSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

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 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 Roles of the Non-Structural Proteins of Influenza A Virus

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 812
Author(s):  
Wenzhuo Hao ◽  
Lingyan Wang ◽  
Shitao Li
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Virus ◽  
Critical Role ◽  
Viral Proteins ◽  
Host Cells ◽  
Structural Proteins ◽  
Host Interactions ◽  
New Findings ◽  
Seasonal Epidemics

Influenza A virus (IAV) is a segmented, negative single-stranded RNA virus that causes seasonal epidemics and has a potential for pandemics. Several viral proteins are not packed in the IAV viral particle and only expressed in the infected host cells. These proteins are named non-structural proteins (NSPs), including NS1, PB1-F2 and PA-X. They play a versatile role in the viral life cycle by modulating viral replication and transcription. More importantly, they also play a critical role in the evasion of the surveillance of host defense and viral pathogenicity by inducing apoptosis, perturbing innate immunity, and exacerbating inflammation. Here, we review the recent advances of these NSPs and how the new findings deepen our understanding of IAV–host interactions and viral pathogenesis.

scholarly journals Acidity/Alkalinity of Japanese Encephalitis Virus E Protein Residue 138 Alters Neurovirulence in Mice

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Xuchen Zheng ◽  
Hao Zheng ◽  
Wu Tong ◽  
Guoxin Li ◽  
Tao Wang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Entry ◽  
Critical Role ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Rational Development ◽  
E Protein

ABSTRACT The Japanese encephalitis virus (JEV) envelope (E) protein, as one of mediators of virus entry into host cells, plays a critical role in determining virulence. The Glu-to-Lys mutation of residue 138 in E protein (E138) plays an important role in attenuating JEV vaccine strain SA14-14-2. However, it is not clear how E138 attenuates JEV. Here, we demonstrate that the Glu-to-Arg mutation of E138 also determines the attenuation of JEV strain 10S3. Likewise, for its parent strain (HEN0701), a virulence strain, the mutations of E138 are responsible for virulence alteration. Furthermore, we demonstrated that mutations of alkaline residues in E138 contributed to the attenuation of neurovirulence; in contrast, mutations of acidic residues enhanced the neurovirulence of the strains. Moreover, acidity in residue E47 had a similar effect on neurovirulence. Furthermore, the alkaline E138 residue enhanced susceptibility to heparin inhibition in vitro and limited JEV diffusion in mouse brain. These results suggest that the acidity/alkalinity of the E138 residue plays an important role in neurovirulence determination. IMPORTANCE The E protein is the only glycoprotein in mature JEV, and it plays an important role in viral neurovirulence. E protein mutations attenuate JEV neurovirulence through unclear mechanisms. Here, we discovered that E138 is a predominant determinant of JEV neurovirulence. We demonstrated that the alkalinity/acidity of E138 determines JEV neurovirulence. These data contribute to the characterization of the E protein and the rational development of novel JEV vaccines.

scholarly journals Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative

2004 ◽  
Vol 78 (19) ◽  
pp. 10803-10813 ◽  
Author(s):  
Barbara Müller ◽  
Jessica Daecke ◽  
Oliver T. Fackler ◽  
Matthias T. Dittmar ◽  
Hanswalter Zentgraf ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Live Cells ◽  
Structural Protein ◽  
Initial Finding ◽  
Particle Assembly ◽  
Immunodeficiency Virus

ABSTRACT The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.

scholarly journals LYT1 Protein Is Required for Efficient In Vitro Infection by Trypanosoma cruzi

2001 ◽  
Vol 69 (6) ◽  
pp. 3916-3923 ◽  
Author(s):  
Rebeca Manning-Cela ◽  
Arantxa Cortés ◽  
Elena González-Rey ◽  
Wesley C. Van Voorhis ◽  
John Swindle ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Mutational Analysis ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Host Cells ◽  
Wild Type ◽  
Stage Transition

ABSTRACT Trypanosoma cruzi invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of theLYT1 gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of LYT1 resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of LYT1 restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments withLYT1-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.

scholarly journals Sensitive detection of LiveEscherichia coliby bacteriophage amplification-coupled immunoassay on the Luminex®MAGPIX instrument

2018 ◽  
Author(s):  
Tomotaka Mido ◽  
Eric M. Schaffer ◽  
Robert W. Dorsey ◽  
Shanmuga Sozhamannan ◽  
E. Randal Hofmann
Keyword(s):  
Plaque Assay ◽  
Simultaneous Detection ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Cell Detection ◽  
Testing Time ◽  
Bacterial Inactivation ◽  
Progeny Phage ◽  
Coupled Assay

AbstractPhages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples. This approach reduces testing time and enhances sensitivity to identify pathogens compared to traditional overnight plaque assay. Further, the assay has the ability to discriminate between live and dead cells since phages require live host cells to infect and replicate. To demonstrate its utility, phage MS2 amplification-coupled, bead-based sandwich type immunoassay on the Luminex®MAGPIX instrument forEscherichia colidetection was performed. The assay not only showed live cell discrimination ability but also a limit ofE. colidetection of 1×102cells/mL of live cells after a 3-hour incubation. In addition, the sensitivity of the assay was not impaired in the presence of dead cells. These results demonstrate that bacteriophage amplification-coupled assay can be a rapid live cell detection assay compared to traditional culture methods and a promising tool for quick validation of bacterial inactivation. Combined with the unique multiplex bead chemistry afforded by Luminex®MAGPIX platform, the phage assay can be expanded to be an ultra-deep multiplex assay for the simultaneous detection of multiple pathogens using specific phages directed against the target pathogens.

scholarly journals Roles of Non-Structural Protein 4A in Flavivirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2077
Author(s):  
Paeka Klaitong ◽  
Duncan R. Smith
Keyword(s):  
Viral Replication ◽  
Critical Role ◽  
Public Health Problem ◽  
Multiple Roles ◽  
Host Cells ◽  
Interferon Response ◽  
Structural Protein ◽  
Comprehensive Overview ◽  
Direct Role ◽  
Developmental Defects

Infections with viruses in the genus Flavivirus are a worldwide public health problem. These enveloped, positive sense single stranded RNA viruses use a small complement of only 10 encoded proteins and the RNA genome itself to remodel host cells to achieve conditions favoring viral replication. A consequence of the limited viral armamentarium is that each protein exerts multiple cellular effects, in addition to any direct role in viral replication. The viruses encode four non-structural (NS) small transmembrane proteins (NS2A, NS2B, NS4A and NS4B) which collectively remain rather poorly characterized. NS4A is a 16kDa membrane associated protein and recent studies have shown that this protein plays multiple roles, including in membrane remodeling, antagonism of the host cell interferon response, and in the induction of autophagy, in addition to playing a role in viral replication. Perhaps most importantly, NS4A has been implicated as playing a critical role in fetal developmental defects seen as a consequence of Zika virus infection during pregnancy. This review provides a comprehensive overview of the multiple roles of this small but pivotal protein in mediating the pathobiology of flaviviral infections.

Sign in / Sign up

Export Citation Format

Share Document