scholarly journals Cell Entry-Independent Role for the Reovirus μ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Katherine E. Roebke ◽  
Pranav Danthi
Keyword(s):  
Cell Death ◽  
Capsid Protein ◽  
Apoptotic Cell ◽  
Guanidine Hydrochloride ◽  
Viral Gene ◽  
Gene Products ◽  
Minus Strand ◽  
Outer Capsid Protein ◽  
Infected Cells ◽  
Outer Capsid

ABSTRACTThe reovirus outer capsid protein μ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized μ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized μ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of μ1 also affects aspects of viral replication. We found that, while μ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the μ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when μ1 is knocked down. Following μ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of μ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to μ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCEWe used mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for the reovirus outer capsid protein μ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection; μ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

scholarly journals Cell entry-independent role for the reovirus μ1 protein in regulating necroptosis and the accumulation of viral gene products

2019 ◽  
Author(s):  
Katherine E Roebke ◽  
Pranav Danthi
Keyword(s):  
Cell Death ◽  
Capsid Protein ◽  
Apoptotic Cell ◽  
Viral Gene ◽  
Gene Products ◽  
Minus Strand ◽  
Virus Infections ◽  
Outer Capsid Protein ◽  
Infected Cells ◽  
Outer Capsid

ABSTRACTThe reovirus outer capsid protein μ1 regulates cell death in infected cells. To distinguish between the role of incoming, capsid-associated and newly synthesized μ1, we used siRNA-mediated knockdown. Loss of newly synthesized μ1 protein does not impact apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of μ1 also impacts aspects of viral replication. We found that while μ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus strand RNA, plus strand RNA, and proteins that are not targeted by the μ1 siRNA accumulate to a greater extent when compared to control siRNA-treated cells. Furthermore, we observe a decrease in sensitivity of these viral products to inhibition by GuHCl (which targets minus strand synthesis to produce dsRNA) when μ1 is knocked down. Following μ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of μ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to a μ1 knockdown potentiates RIP3 dependent cell death.IMPORTANCEWe use mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for reovirus outer capsid protein μ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection. μ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

scholarly journals Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Katherine E. Roebke ◽  
Yingying Guo ◽  
John S. L. Parker ◽  
Pranav Danthi
Keyword(s):  
Cell Death ◽  
Capsid Protein ◽  
Rna Synthesis ◽  
Innate Immune ◽  
Type I ◽  
Outer Capsid Protein ◽  
Immune Signaling ◽  
Innate Immune Signaling ◽  
Dsrna Binding ◽  
Outer Capsid

ABSTRACT Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic double-stranded RNA (dsRNA). The reovirus outer capsid protein μ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with μ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of μ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity. IMPORTANCE We use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here, we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

scholarly journals Reovirus σ3 protein limits interferon expression and cell death induction

2020 ◽  
Author(s):  
Katherine E Roebke ◽  
Yingying Guo ◽  
John S. L. Parker ◽  
Pranav Danthi
Keyword(s):  
Cell Death ◽  
Capsid Protein ◽  
Rna Synthesis ◽  
Innate Immune ◽  
Type I ◽  
Outer Capsid Protein ◽  
Immune Signaling ◽  
Innate Immune Signaling ◽  
Dsrna Binding ◽  
Outer Capsid

ABSTRACTInduction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic dsRNA. The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating cell death, we used siRNA-mediated knockdown. Similar to that observed by diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. σ3 knockdown does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or cell death. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and cell death following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCEWe use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. σ3 limits the induction of necroptosis by preventing excessive production of interferon following infection.

Development and efficacy of a grass carp reovirus (GCRV) outer capsid protein VP7 subunit vaccine

2012 ◽  
Vol 37 (6) ◽  
pp. 659-664 ◽  
Author(s):  
Shi-ying XU ◽  
Jing-hui LI ◽  
Yong ZOU ◽  
Lin LIU ◽  
Cheng-liang GONG ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Grass Carp ◽  
Subunit Vaccine ◽  
Outer Capsid Protein ◽  
Grass Carp Reovirus ◽  
Outer Capsid

scholarly journals Chinese Giant Salamander (Andrias davidianus) Iridovirus Infection Leads to Apoptotic Cell Death through Mitochondrial Damage, Caspases Activation, and Expression of Apoptotic-Related Genes

2019 ◽  
Vol 20 (24) ◽  
pp. 6149 ◽  
Author(s):  
Yiqun Li ◽  
Nan Jiang ◽  
Yuding Fan ◽  
Yong Zhou ◽  
Wenzhi Liu ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Mitochondrial Damage ◽  
Apoptotic Cell Death ◽  
Economic Losses ◽  
P53 Expression ◽  
Andrias Davidianus ◽  
Chinese Giant Salamander ◽  
Infected Cells ◽  
Apoptotic Genes

Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander (Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation.

scholarly journals Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

2011 ◽  
Vol 85 (16) ◽  
pp. 8141-8148 ◽  
Author(s):  
A. Fokine ◽  
M. Z. Islam ◽  
Z. Zhang ◽  
V. D. Bowman ◽  
V. B. Rao ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Outer Capsid Protein ◽  
Outer Capsid

scholarly journals Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-Å resolution and its relevance to the assembly of icosahedral viruses

2017 ◽  
Vol 114 (39) ◽  
pp. E8184-E8193 ◽  
Author(s):  
Zhenguo Chen ◽  
Lei Sun ◽  
Zhihong Zhang ◽  
Andrei Fokine ◽  
Victor Padilla-Sanchez ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Bacteriophage T4 ◽  
Hexagonal Lattice ◽  
Scaffolding Proteins ◽  
Outer Capsid Protein ◽  
Head Assembly ◽  
Icosahedral Viruses ◽  
End Caps ◽  
Outer Capsid

The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend= 13 for end caps and Tmid= 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

scholarly journals Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

2004 ◽  
Vol 78 (16) ◽  
pp. 8732-8745 ◽  
Author(s):  
Amy L. Odegard ◽  
Kartik Chandran ◽  
Xing Zhang ◽  
John S. L. Parker ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Structural Changes ◽  
General Mechanism ◽  
Membrane Penetration ◽  
Outer Capsid Protein ◽  
Core Proteins ◽  
Peptide Release ◽  
Outer Capsid

ABSTRACT Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

scholarly journals Activation and Antagonism of the OAS–RNase L Pathway

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 14
Author(s):  
Susan R. Weiss
Keyword(s):  
Cell Death ◽  
Antiviral Activity ◽  
Apoptotic Cell ◽  
Early Response ◽  
Apoptotic Cell Death ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Viral Genomes ◽  
L System ◽  
Infected Cells

The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, virus replication and spread, leading to apoptotic cell death. Endogenous host dsRNA, which accumulates in the absence of adenosine deaminase acting on RNA (ADAR)1, can also activate RNase L and lead to apoptotic cell death. RNase L activation and antiviral activity during infections with several types of viruses in human and bat cells is dependent on OAS3 but independent of virus-induced interferon (IFN) and, thus, RNase L can be activated even in the presence of IFN antagonists. Differently from other human viruses examined, Zika virus is resistant to the antiviral activity of RNase L and instead utilizes RNase L to enhance its replication factories to produce more infectious virus. Some betacoronaviruses antagonize RNase L activation by expressing 2′,5′-phosphodiesterases (PDEs) that cleave 2-5A and thereby antagonize activation of RNase L. The best characterized of these PDEs is the murine coronavirus (MHV) NS2 accessory protein. Enzymatically active NS2 is required for replication in myeloid cells and in the liver. Interestingly, while wild type mice clear MHV from the liver by 7–10 days post-infection, RNase L knockout mice fail to effectively clear MHV, probably due to diminished apoptotic death of infected cells. We suggest that RNase L antiviral activity stems from direct cleavage of viral genomes and cessation of protein synthesis as well as through promoting death of infected cells, limiting the spread of virus. Importantly, OASs are pattern recognition receptors and the OAS–RNase L pathway is a primary innate response pathway to viruses, capable of early response, coming into play before IFN is induced or when the virus shuts down IFN signaling.

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