Double-Stranded RNA Replication in Yeast: The Killer System

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.

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2P261 Double-stranded RNA formation during Qβ long RNA replication(20. Origin of life & Evolution,Poster)

Seibutsu Butsuri ◽

10.2142/biophys.53.s202_2 ◽

2013 ◽

Vol 53 (supplement1-2) ◽

pp. S202

Author(s):

Kimihito Usui ◽

Norikazu Ichihashi ◽

Yasuaki Kazuta ◽

Tetsuya Yomo

Keyword(s):

Origin Of Life ◽

Rna Replication ◽

Double Stranded Rna ◽

Rna Formation

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K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4807 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4807-4815 ◽

Cited By ~ 61

Author(s):

M J Schmitt ◽

D J Tipper

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytoplasmic Membrane ◽

Yeast Cells ◽

Cross Hybridization ◽

Double Stranded Rna ◽

Killer System ◽

Dsrna Viruses ◽

Viruslike Particles ◽

Killer Virus

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Journal of Virology ◽

10.1128/jvi.03080-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3676-3683 ◽

Cited By ~ 6

Author(s):

James R. Short ◽

Jeffrey A. Speir ◽

Radhika Gopal ◽

Logan M. Pankratz ◽

Jason Lanman ◽

...

Keyword(s):

Host Defense ◽

Rna Synthesis ◽

Molecular Mechanisms ◽

Rna Viruses ◽

Rna Replication ◽

Viral Rna ◽

Flock House Virus ◽

Double Stranded Rna ◽

Content Type ◽

Positive Sense

ABSTRACTViruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infectsDrosophilacells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infectedDrosophilacells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infectedDrosophilacells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

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The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast

PLoS Pathogens ◽

10.1371/journal.ppat.1006520 ◽

2017 ◽

Vol 13 (7) ◽

pp. e1006520 ◽

Cited By ~ 18

Author(s):

Nikolay Kovalev ◽

Jun-ichi Inaba ◽

Zhenghe Li ◽

Peter D. Nagy

Keyword(s):

Rna Replication ◽

Double Stranded Rna ◽

Replication Intermediate

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Fate of Minus-Strand Templates and Replication Complexes Produced by a P23-Cleavage-Defective Mutant of Sindbis Virus

Journal of Virology ◽

10.1128/jvi.00056-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8553-8564 ◽

Cited By ~ 11

Author(s):

Junbo Mai ◽

Stanley G. Sawicki ◽

Dorothea L. Sawicki

Keyword(s):

Sindbis Virus ◽

Rna Replication ◽

The Novel ◽

Minus Strand ◽

Wild Type ◽

Double Stranded Rna ◽

Normal Functions ◽

Defective Mutant ◽

Infected Cells ◽

The Stability

ABSTRACT SIN2V is an engineered mutant Sindbis virus (SIN) that is unable to process the P23 cleavage site in polyproteins P123 and P1234 that are translated from the genome after its entry into cells. Unlike wild-type (wt) SIN, it caused minus strands to be made continuously and replication-transcription complex (RTC) activity to be unstable (R. Gorchakov, E. Frolova, S. Sawicki, S. Atasheva, D. Sawicki, and I. Frolov, J. Virol. 82:6218-6231, 2008). We examined further the effects of P23 on SIN RNA replication and RTC activity. Continuous minus-strand synthesis by SIN2V produced 250% of wt levels of minus strands but accumulated only 110% of wt levels (0.39 pg, or 2.7 × 104 molecules of double-stranded RNA per cell). Because SIN2V-infected cells accumulated only 40% of the minus strands that were made, cells must possess some process to limit RTC accumulation. The loss of activity by SIN2V RTC after translation was inhibited was stochastic and not due to their inherent instability, based on finding that activity was lost without the degradation of the minus-strand templates. In addition to their normal functions, P23 RTCs exhibited the novel phenotype of being unable to switch from making less to making more genomes than subgenomic 26S mRNA at late times during infections. Our results lend credence to the hypothesis that nsP2 (and possibly nsP3) possesses functions other than those needed solely for RTC activity and that it may also act with the host to regulate minus-strand synthesis and the stability of the RTC.

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K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4807-4815.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4807-4815

Author(s):

M J Schmitt ◽

D J Tipper

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytoplasmic Membrane ◽

Yeast Cells ◽

Cross Hybridization ◽

Double Stranded Rna ◽

Killer System ◽

Dsrna Viruses ◽

Viruslike Particles ◽

Killer Virus

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

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Double-stranded RNA induces similar pulmonary dysfunction to respiratory syncytial virus in BALB/c mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00398.2010 ◽

2011 ◽

Vol 301 (1) ◽

pp. L99-L109 ◽

Cited By ~ 35

Author(s):

Famke Aeffner ◽

Zachary P. Traylor ◽

Erin N. Z. Yu ◽

Ian C. Davis

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Purinergic Receptor ◽

Rna Replication ◽

Poly I:C ◽

Lung Edema ◽

Adrenergic Agonist ◽

Double Stranded Rna ◽

Syncytial Virus ◽

Replication Intermediates

Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl− transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3−/− mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2–3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl− secretion in the lung or severe, lethal hypoxemia, both of which are features of influenza infection.

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