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Paul Ahlquist - One of the best experts on this subject based on the ideXlab platform.
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role of host reticulon proteins in rearranging membranes for positive strand RNA Virus replication
Current Opinion in Microbiology, 2012Co-Authors: Arturo Diaz, Paul AhlquistAbstract:Positive-Strand RNA [(+)RNA] Viruses are responsible for numerous human, animal, and plant diseases. Because of the limiting coding capacity of (+)RNA Viruses, their replication requires a complex orchestration of interactions between the viral genome, viral proteins and exploited host factors. To replicate their genomic RNAs, (+)RNA Viruses induce membrane rearrangements that create membrane-linked RNA replication compartments. Along with substantial advances on the ultrastructure of the membrane-bound RNA replication compartments, recent results have shed light into the role that host factors play in rearranging these membranes. This review focuses on recent insights that have driven a new understanding of the role that the membrane-shaping host reticulon homology domain proteins (RHPs) play in facilitating the replication of various (+)RNA Viruses.
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host acyl coenzyme a binding protein regulates replication complex assembly and activity of a positive strand RNA Virus
Journal of Virology, 2012Co-Authors: Jiantao Zhang, Paul Ahlquist, Arturo Diaz, Lan Mao, Xiaofeng WangAbstract:All Positive-Strand RNA Viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic Virus (BMV) induces two alteRNAte forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.
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cytoplasmic viral replication complexes
Cell Host & Microbe, 2010Co-Authors: Johan Den A Boon, Paul Ahlquist, Arturo DiazAbstract:Many Viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse Positive-Strand RNA Viruses have further elucidated the ultrastructure of membrane-bound RNA replication complexes and how these complexes function in close coordination with virion assembly and budding. The structure, function, and assembly of some Positive-Strand RNA Virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA Virus and retroVirus virions and more general similarities with the replication factories of cytoplasmic DNA Viruses.
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Host factors in Positive-Strand RNA Virus genome replication.
Journal of Virology, 2003Co-Authors: Paul Ahlquist, Amine Noueiry, David B. KushnerAbstract:All Viruses are gene poor relative to their hosts: even the largest viral genomes only encode hundreds of genes, while those of host cells typically encode tens of thousands of genes. Thus, most steps in Virus infection involve interactions between relatively few different types of viral components and much more complex pools of host factors. This sea of host factors represents both the essential milieu to which Viruses must adapt for survival and a tremendous, manipulatable resource for gene-poor Viruses. Accordingly, host factors play important roles in most steps of viral infection, and identifying such host factors and their contributions has long been recognized as an important frontier. The continuing emergence of close integration between viral and host functions in infection suggests moving beyond separate views of Virus and host to a more holistic view of the Virus-infected cell as a unified entity that constitutes the functional unit of infection. One area in which the importance of host factors is increasingly emerging is the replication of Positive-Strand RNA Viruses. Positive-Strand RNA Viruses encompass over one-third of all Virus genera and include numerous pathogens, such as the severe acute respiratory syndrome coronaVirus SARS, hepatitis C Virus (HCV), and many of the Viruses on the U.S. Health and Human Services Department Select List of potential bioterrorism agents. Host factors participate in most, if not all, steps of Positive-Strand RNA Virus infection, including entry, viral gene expression, virion assembly, and release. Moreover, host factors are targeted by Positive-Strand RNA Viruses to modulate host gene expression and defenses. This review focuses on host factors involved in positivestrand RNA Virus genome replication. The evidence for such host factor involvement has come from varied genetic and biochemical approaches (15). These include, among others, studies based on the varying permissiveness of some cell types and extracts for RNA replication (2, 3, 4, 19); identification of many host proteins that interact with viral genomic RNAs or replication proteins and, in some cases, have been functionally linked to replication (5, 18, 27, 35, 37, 40); and mutational screens in genetic model systems, such as Arabidopsis thaliana (17, 39) and the yeast Saccharomyces cerevisiae (8, 11, 16, 23, 38). Recent data show that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral RNA replication templates, activating the complex for RNA synthesis, and other steps. Each of these Virushost interactions may contribute to the host specificity, tissue specificity, or pathology of infections. Each such Virus-host interaction also represents a potential target for Virus control or for optimization to improve beneficial uses of Viruses and their components. Positive-Strand RNA Viruses can be divided into a number of superfamilies defined by distinguishable RNA replication genes and features (41). Nevertheless, the RNA replication mechanisms of these Viruses share sufficient similarities to make it reasonable to discuss their replication as a class and to attempt to draw general lessons by comparing examples from different superfamilies. As discussed further below, a few of the common features shared by Positive-Strand RNA Viruses are the need to coordinate use of the infecting viral genomic RNA as a template for translation and replication, assembly of replication complexes on intracellular membranes, and production of 10- to 100-fold excesses of positive- over negativestrand RNA. A simplified general scheme for RNA replication by Positive-Strand RNA Viruses is shown in Fig. 1. Below we discuss the involvement of host factors in the various stages of this RNA replication process, using examples from a number of Viruses. We regret that space limitations do not allow us to cite all of the work being done in this exciting area.
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a brome mosaic Virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo
Journal of Virology, 1999Co-Authors: Michael L Sullivan, Paul AhlquistAbstract:Brome mosaic Virus (BMV), a Positive-Strand RNA Virus in the alphaVirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous β-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and Positive-Strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.
Yoshifumi Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of solenopsis invicta Virus 3 a new positive strand RNA Virus infecting the red imported fire ant solenopsis invicta
Virology, 2009Co-Authors: Steven M Valles, Yoshifumi HashimotoAbstract:Abstract We report the discovery of a new Virus from the red imported fire ant, Solenopsis invicta . Solenopsis invicta Virus 3 (SINV-3) represents the third Virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584 ), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroViruses, iflaViruses, plant small RNA Viruses, picoRNAViruses, and 4 unassigned Positive-Strand RNA Viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly Virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the Virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.
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a new positive strand RNA Virus with unique genome characteristics from the red imported fire ant solenopsis invicta
Virology, 2007Co-Authors: Steven M Valles, Charles A Strong, Yoshifumi HashimotoAbstract:We report the discovery of a new Virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This Virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta Virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5' RACE reactions, a 3' RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3' end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of > or =100 codons) and 5 minor ORFs (comprised of 50-99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a Positive-Strand RNA Virus. Further evidence for this conclusion includes abolished RT-PCR amplification by RNAse treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of Positive-Strand RNA Viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaViruses, PicoRNAviridae, and plant small RNA Viruses.
Steven M Valles - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of solenopsis invicta Virus 3 a new positive strand RNA Virus infecting the red imported fire ant solenopsis invicta
Virology, 2009Co-Authors: Steven M Valles, Yoshifumi HashimotoAbstract:Abstract We report the discovery of a new Virus from the red imported fire ant, Solenopsis invicta . Solenopsis invicta Virus 3 (SINV-3) represents the third Virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584 ), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroViruses, iflaViruses, plant small RNA Viruses, picoRNAViruses, and 4 unassigned Positive-Strand RNA Viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly Virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the Virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.
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a new positive strand RNA Virus with unique genome characteristics from the red imported fire ant solenopsis invicta
Virology, 2007Co-Authors: Steven M Valles, Charles A Strong, Yoshifumi HashimotoAbstract:We report the discovery of a new Virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This Virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta Virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5' RACE reactions, a 3' RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3' end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of > or =100 codons) and 5 minor ORFs (comprised of 50-99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a Positive-Strand RNA Virus. Further evidence for this conclusion includes abolished RT-PCR amplification by RNAse treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of Positive-Strand RNA Viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaViruses, PicoRNAviridae, and plant small RNA Viruses.
Isabelle Jupin - One of the best experts on this subject based on the ideXlab platform.
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A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity
The EMBO Journal, 2011Co-Authors: Mélanie Chenon, Soizic Cheminant, Laurent Camborde, Isabelle JupinAbstract:Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host–pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the Positive-Strand RNA Virus Turnip yellow mosaic Virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein—its binding partner within replication complexes—leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.
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A Turnip yellow mosaic Virus infection system in Arabidopsis suspension cell culture
FEBS Letters, 2007Co-Authors: Laurent Camborde, Mildred Noizet, Vincent Tournier, Isabelle JupinAbstract:Turnip yellow mosaic Virus (TYMV) is a Positive-Strand RNA Virus able to infect Arabidopsis thaliana. To establish a TYMV infection system in Arabidopsis cell culture, TYMV replicons with the capsid protein gene replaced by a reporter gene expressing the Sh ble protein conferring zeocin resistance were used to transfect Arabidopsis cells. Zeocin-resistant Arabidopsis calli were used to generate a suspension cell culture. Detection of viral proteins and RNAs after 18 months in culture demonstrated persistent replication of the replicon. The Arabidopsis cell culture yielded soluble, active replication complexes, providing a useful tool to study host factors involved in TYMV replication.
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targeting of the turnip yellow mosaic Virus 66k replication protein to the chloroplast envelope is mediated by the 140k protein
Journal of Virology, 2003Co-Authors: Delphine Prodhomme, Vincent Tournier, Anna Jakubiec, Gabriele Drugeon, Isabelle JupinAbstract:Turnip yellow mosaic Virus (TYMV), a Positive-Strand RNA Virus in the alphaVirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to Virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.
David J. Miller - One of the best experts on this subject based on the ideXlab platform.
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complementary transcriptomic lipidomic and targeted functional genetic analyses in cultured drosophila cells highlight the role of glycerophospholipid metabolism in flock house Virus RNA replication
BMC Genomics, 2010Co-Authors: Kathryn M Castorena, Kenneth A Stapleford, David J. MillerAbstract:Background Cellular membranes are crucial host components utilized by Positive-Strand RNA Viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of Positive-Strand RNA Virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with Positive-Strand RNA Virus replication been examined in detail.
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Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House Virus RNA replication
BMC Genomics, 2010Co-Authors: Kathryn M Castorena, Kenneth A Stapleford, David J. MillerAbstract:Cellular membranes are crucial host components utilized by Positive-Strand RNA Viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of Positive-Strand RNA Virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with Positive-Strand RNA Virus replication been examined in detail. In this study we used parallel and complementary global and targeted approaches to examine the impact of lipid metabolism on the replication of the well-studied model alphanodaVirus Flock House Virus (FHV). We found that FHV RNA replication in cultured Drosophila S2 cells stimulated the transcriptional upregulation of several lipid metabolism genes, and was also associated with increased phosphatidylcholine accumulation with preferential increases in lipid molecules with longer and unsaturated acyl chains. Furthermore, targeted RNA interference-mediated downregulation of candidate glycerophospholipid metabolism genes revealed a functional role of several genes in Virus replication. In particular, we found that downregulation of Cct1 or Cct2, which encode essential enzymes for phosphatidylcholine biosynthesis, suppressed FHV RNA replication. These results indicate that glycerophospholipid metabolism, and in particular phosphatidylcholine biosynthesis, plays an important role in FHV RNA replication. Furthermore, they provide a framework in which to further explore the impact of specific steps in lipid metabolism on FHV replication, and potentially identify novel cellular targets for the development of drugs to inhibit Positive-Strand RNA Viruses.
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Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells
Journal of Virology, 2001Co-Authors: David J. Miller, Michael SchwartzAbstract:The identification and characterization of host cell membranes essential for Positive-Strand RNA Virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodaVirus flock house Virus (FHV) is a Positive-Strand RNA Virus with one of the smallest known genomes among animal RNA Viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other Positive-Strand RNA Viruses. Confocal immunofluorescence microscopy and Virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusVirus- and togaVirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of Positive-Strand RNA Viruses from other families.