scholarly journals MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

Cell Reports ◽  
2012 ◽  
Vol 2 (5) ◽  
pp. 1187-1196 ◽  
Author(s):  
Qian Feng ◽  
Stanleyson V. Hato ◽  
Martijn A. Langereis ◽  
Jan Zoll ◽  
Richard Virgen-Slane ◽  
...  
Keyword(s):  
Replicative Form ◽  
Double Stranded Rna ◽  
Infected Cells

scholarly journals The Leader Protein of Theiler's Virus Prevents the Activation of PKR

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Teresa Cesaro ◽  
Thomas Michiels
Keyword(s):  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Granule Formation ◽  
Double Stranded Rna ◽  
Eif2α Phosphorylation ◽  
Leader Protein ◽  
Infected Cells ◽  
L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

scholarly journals Activation of MDA5 Requires Higher-Order RNA Structures Generated during Virus Infection

2009 ◽  
Vol 83 (20) ◽  
pp. 10761-10769 ◽  
Author(s):  
Andreas Pichlmair ◽  
Oliver Schulz ◽  
Choon-Ping Tan ◽  
Jan Rehwinkel ◽  
Hiroki Kato ◽  
...  
Keyword(s):  
Higher Order ◽  
Encephalomyocarditis Virus ◽  
Signaling Cascade ◽  
Rna Structures ◽  
Innate Response ◽  
Double Stranded Rna ◽  
Infected Cells ◽  
Alpha Beta ◽  
Complementary Rnas ◽  
Inducible Gene

ABSTRACT Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-α/β) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-α/β production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-α/β production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Caitlin A. O'Brien ◽  
Jessica J. Harrison ◽  
Agathe M. G. Colmant ◽  
Renee J. Traves ◽  
Devina Paramitha ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Untranslated Regions ◽  
Fixation Method ◽  
Enveloped Viruses ◽  
Double Stranded Rna ◽  
Vertebrate Cell ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Dsrna Viruses ◽  
Arboviral Disease

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

scholarly journals Lassa Virus, but Not Highly Pathogenic New World Arenaviruses, Restricts Immunostimulatory Double-Stranded RNA Accumulation during Infection

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Elizabeth J. Mateer ◽  
Junki Maruyama ◽  
Galen E. Card ◽  
Slobodan Paessler ◽  
Cheng Huang
Keyword(s):  
Immune Responses ◽  
Innate Immune ◽  
Lassa Virus ◽  
Innate Immune Responses ◽  
Highly Pathogenic ◽  
Double Stranded Rna ◽  
L Protein ◽  
Biochemical Assays ◽  
Infected Cells ◽  
First Time

ABSTRACT The arenaviruses Lassa virus (LASV), Junín virus (JUNV), and Machupo virus (MACV) can cause severe and fatal diseases in humans. Although these pathogens are closely related, the host immune responses to these virus infections differ remarkably, with direct implications for viral pathogenesis. LASV infection is immunosuppressive, with a very low-level interferon response. In contrast, JUNV and MACV infections stimulate a robust interferon (IFN) response in a retinoic acid-inducible gene I (RIG-I)-dependent manner and readily activate protein kinase R (PKR), a known host double-stranded RNA (dsRNA) sensor. In response to infection with RNA viruses, host nonself RNA sensors recognize virus-derived dsRNA as danger signals and initiate innate immune responses. Arenavirus nucleoproteins (NPs) contain a highly conserved exoribonuclease (ExoN) motif, through which LASV NP has been shown to degrade virus-derived immunostimulatory dsRNA in biochemical assays. In this study, we for the first time present evidence that LASV restricts dsRNA accumulation during infection. Although JUNV and MACV NPs also have the ExoN motif, dsRNA readily accumulated in infected cells and often colocalized with dsRNA sensors. Moreover, LASV coinfection diminished the accumulation of dsRNA and the IFN response in JUNV-infected cells. The disruption of LASV NP ExoN with a mutation led to dsRNA accumulation and impaired LASV replication in minigenome systems. Importantly, both LASV NP and RNA polymerase L protein were required to diminish the accumulation of dsRNA and the IFN response in JUNV infection. For the first time, we discovered a collaboration between LASV NP ExoN and L protein in limiting dsRNA accumulation. Our new findings provide mechanistic insights into the differential host innate immune responses to highly pathogenic arenavirus infections. IMPORTANCE Arenavirus NPs contain a highly conserved DEDDh ExoN motif, through which LASV NP degrades virus-derived, immunostimulatory dsRNA in biochemical assays to eliminate the danger signal and inhibit the innate immune response. Nevertheless, the function of NP ExoN in arenavirus infection remains to be defined. In this study, we discovered that LASV potently restricts dsRNA accumulation during infection and minigenome replication. In contrast, although the NPs of JUNV and MACV also harbor the ExoN motif, dsRNA readily formed during JUNV and MACV infections, accompanied by IFN and PKR responses. Interestingly, LASV NP alone was not sufficient to limit dsRNA accumulation. Instead, both LASV NP and L protein were required to restrict immunostimulatory dsRNA accumulation. Our findings provide novel and important insights into the mechanism for the distinct innate immune response to these highly pathogenic arenaviruses and open new directions for future studies.

scholarly journals Visualization of Double-Stranded RNA in Cells Supporting Hepatitis C Virus RNA Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2182-2195 ◽  
Author(s):  
Paul Targett-Adams ◽  
Steeve Boulant ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Three Dimensional ◽  
Rna Replication ◽  
Hcv Rna ◽  
Double Stranded Rna ◽  
Core Proteins ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.

Die Bildung von RNS-Doppelstrang zur Vermehrung eines RNS enthaltenden Bakteriophagen

1964 ◽  
Vol 19 (7) ◽  
pp. 593-604 ◽  
Author(s):  
Hans Christian Kaerner ◽  
Hartmut Hoffmann-Berling
Keyword(s):  
Molecular Weight ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Radioactive Material ◽  
Double Stranded Rna ◽  
Infected Cells ◽  
Rna Phage ◽  
Denaturation Profile ◽  
Molecular Weight Form ◽  
Latent Phase

The RNA phage fr induces in Escherichia coli cells the production of double stranded RNA, which is identified by its thermal denaturation profile ( Tm 101 °C in 0,2-m. Na⊕ ), by its nonreactivity with formaldehyde and by its buoyant density in Cs2SO4 (1,609 g cm-3 , compared to that of fr-RNA = 1,634 g cm-3 ). Unless denatured the double strand is resistant to RNase. In its high molecular weight form the double stranded R N A has twice the weight of fr-RNA, as estimated from the sedimentation coefficient (s20 = 14,5). The base ratios are those expected for a double stranded replicative from of fr-RNA. By melting and annealing one of the strands of the non-radioactive material can be exchanged for 32P-fr-RNA from phage particles. Infectiosity of the doublestranded RNAhas not yet been shown. Extracts from infected cells contain double strand bound to the 30 - 50 s fraction; there is also double strand in the supernatant, apparently in the form of relatively low molecular weight fragments. The double stranded RNA, isolated at the height of infection, accounts for 3 - 8% of the cellular RNA. Cells infected with 32P-fr show a surprisingly large part of the infecting RNA bound to ribosomes quite late in the latent phase. The meaning of this result is discussed.

scholarly journals Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.

1997 ◽  
Vol 71 (3) ◽  
pp. 1992-2003 ◽  
Author(s):  
K V Kibler ◽  
T Shors ◽  
K B Perkins ◽  
C C Zeman ◽  
M P Banaszak ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Double Stranded Rna ◽  
Infected Cells

#51: Neurotropic Astrovirus-VA1: Identifying the distinctions from classical genotypes

2021 ◽  
Vol 10 (Supplement_2) ◽  
pp. S15-S16
Author(s):  
Amy E Davis ◽  
Virginia Hargest ◽  
Shaoyuan Tan ◽  
Qidong Jia ◽  
Valerie Cortez ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Antiviral Drug ◽  
Clinical Importance ◽  
Intestinal Epithelial ◽  
Viral Kinetics ◽  
Double Stranded Rna ◽  
Human Astroviruses ◽  
Inflammatory Cytokine Response ◽  
Infected Cells

Abstract Background Human astroviruses (HAstV) are a leading cause of acute gastroenteritis in children, particularly those under the age of 2 or with immunosuppressive conditions. Indeed, our studies suggest that children with hematological malignancies are at high risk of infection. However, it has become increasingly clear that HAstV infections can also be associated with respiratory and even central nervous system (CNS) diseases. In the last decade, there have been at least 12 cases of astrovirus-induced CNS disease resulting in encephalitis and meningitis, including 2 St Jude patients. The CNS-associated infections are overwhelmingly fatal and are primarily caused by a novel astrovirus genotype, VA1, that is genetically more similar to animal astroviruses that also induce CNS-associated infections. The recent ability to propagate the VA1 strain in cell lines affords us the opportunity to better understand VA1 infection and how it compares to the better-studied HAstV1 strain. Methods Viral kinetics were determined by infection of human intestinal adenocarcinoma Caco-2 cells with either HAstV1 or VA1 and monitored for 24 hours post-infection (hpi). Infected cells were identified via immunofluorescent microscopy using antibodies against double-stranded RNA and viral capsid. Epithelial permeability of astrovirus-infected Caco-2 cells was measured using transepithelial electrical resistance (TER) and monitored for 24hpi. Nitazoxanide efficacy was identified by immunofluorescent analysis as described previously. Results Our findings demonstrate that HAstV1 and VA1 replicate in intestinal epithelial cells without inducing cell death or a pro-inflammatory cytokine response. However, these two strains have distinct replication kinetics. VA1 appears to have an ~8 hour lag in the expression of dsRNA and capsid protein compared to HAstV1 and does not require exogenous proteases to process the viral outer coat (capsid) protein, Additionally, we demonstrated that the increase in epithelial barrier permeability associated with HAstV1-infection is not found in VA1-infected cells, which is intriguing and may explain why HAstV1 is more likely to cause diarrhea. Of clinical importance, we have revealed a similar susceptibility of VA1 to the antiviral drug, nitazoxanide, which we have recently demonstrated its effectiveness inhibiting classical HAstV strains. Conclusion Overall, our studies highlight that VA1 pathogenesis is distinct from HAstV1, which could explain the differences in vivo. Future studies will be necessary to investigate viral replication and pathogenesis in distinct neuronal cells and in vivo.

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