Mitochondrion-Enriched Anionic Phospholipids Facilitate Flock House Virus RNA Polymerase Membrane Association

ABSTRACT One characteristic of all positive-strand RNA viruses is the necessity to assemble viral RNA replication complexes on host intracellular membranes, a process whose molecular details are poorly understood. To study viral replication complex assembly we use the established model system of Flock House virus (FHV), which assembles its replication complexes on the mitochondrial outer membrane. The FHV RNA-dependent RNA polymerase, protein A, is the only viral protein necessary for genome replication in the budding yeast Saccharomyces cerevisiae. To examine the host components involved in protein A-membrane interactions, an initial step of FHV RNA replication complex assembly, we established an in vitro protein A membrane association assay. Protein A translated in vitro rapidly and specifically associated with mitochondria isolated from yeast, insect, and mammalian cells. This process was temperature dependent but independent of protease-sensitive mitochondrial outer membrane components or the host mitochondrial import machinery. Furthermore, lipid-binding studies revealed that protein A preferentially bound to specific anionic phospholipids, in particular the mitochondrion-specific phospholipid cardiolipin. These studies implicate membrane phospholipids as important host determinants for FHV RNA polymerase membrane association and provide evidence for the involvement of host phospholipids in positive-strand RNA virus membrane-specific targeting.

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Flock House Virus RNA Polymerase Is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion

Journal of Virology ◽

10.1128/jvi.76.19.9856-9867.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9856-9867 ◽

Cited By ~ 87

Author(s):

David J. Miller ◽

Paul Ahlquist

Keyword(s):

Transmembrane Domain ◽

Rna Viruses ◽

Protein A ◽

Rna Replication ◽

Membrane Insertion ◽

Membrane Association ◽

Flock House Virus ◽

Mitochondrial Localization ◽

Mitochondrial Membranes ◽

Positive Strand Rna

ABSTRACT Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.

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Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA

Journal of Virology ◽

10.1128/jvi.02267-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4633-4644 ◽

Cited By ~ 34

Author(s):

Priscilla M. Van Wynsberghe ◽

Hau-Ren Chen ◽

Paul Ahlquist

Keyword(s):

Rna Polymerase ◽

Transmembrane Domain ◽

Protein A ◽

Rna Replication ◽

Replication Protein A ◽

Replication Protein ◽

Membrane Association ◽

Genome Replication ◽

Genomic Rna ◽

Positive Strand Rna

ABSTRACT Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (AD692E), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein AD692E stimulated RNA1 accumulation in wild-type cells and in xrn1 − yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.

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An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

Journal of Virology ◽

10.1128/jvi.78.20.11393-11400.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11393-11400 ◽

Cited By ~ 112

Author(s):

Menashe Elazar ◽

Ping Liu ◽

Charles M. Rice ◽

Jeffrey S. Glenn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hcv Rna ◽

Membrane Association ◽

Amphipathic Helix ◽

Nonstructural Proteins ◽

Replication Complex ◽

Cytoplasmic Membranes ◽

Positive Strand Rna

ABSTRACT Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

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5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

Journal of Virology ◽

10.1128/jvi.02040-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 2976-2988 ◽

Cited By ~ 24

Author(s):

Priscilla M. Van Wynsberghe ◽

Paul Ahlquist

Keyword(s):

Complex Formation ◽

Mutational Analysis ◽

Protein A ◽

Rna Replication ◽

Viral Rna ◽

Yeast Cells ◽

Positive Strand ◽

Replication Complex ◽

Negative Strand ◽

Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

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Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

Journal of Virology ◽

10.1128/jvi.78.23.13007-13018.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13007-13018 ◽

Cited By ~ 7

Author(s):

Christopher T. Cornell ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Genetically Modified ◽

Rna Replication ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Wild Type ◽

Positive Strand ◽

Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

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Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Journal of Virology ◽

10.1128/jvi.75.23.11664-11676.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11664-11676 ◽

Cited By ~ 155

Author(s):

David J. Miller ◽

Michael D. Schwartz ◽

Paul Ahlquist

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Protein A ◽

Rna Replication ◽

Yeast Cells ◽

Flock House Virus ◽

Mitochondrial Membranes ◽

Positive Strand ◽

Positive Strand Rna Virus ◽

Positive Strand Rna

ABSTRACT 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 ofDrosophila 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.

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The Cellular Chaperone Heat Shock Protein 90 Facilitates Flock House Virus RNA Replication in Drosophila Cells

Journal of Virology ◽

10.1128/jvi.79.11.6827-6837.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6827-6837 ◽

Cited By ~ 65

Author(s):

Kathryn M. Kampmueller ◽

David J. Miller

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Protein A ◽

Heat Shock Protein 90 ◽

Rna Replication ◽

Viral Rna ◽

Flock House Virus ◽

S2 Cells ◽

Complex Assembly ◽

Replication Complex

ABSTRACT The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.

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Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Journal of Virology ◽

10.1128/jvi.00525-19 ◽

2019 ◽

Vol 93 (19) ◽

Cited By ~ 1

Author(s):

Philipp Schult ◽

Maren Nattermann ◽

Chris Lauber ◽

Stefan Seitz ◽

Volker Lohmann

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Rna Viruses ◽

Rna Replication ◽

Rna Dependent Rna Polymerase ◽

Internal Initiation ◽

Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

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A Functional Heat Shock Protein 90 Chaperone Is Essential for Efficient Flock House Virus RNA Polymerase Synthesis in Drosophila Cells

Journal of Virology ◽

10.1128/jvi.00189-07 ◽

2007 ◽

Vol 81 (16) ◽

pp. 8412-8420 ◽

Cited By ~ 33

Author(s):

Kathryn M. Castorena ◽

Spencer A. Weeks ◽

Kenneth A. Stapleford ◽

Amy M. Cadwallader ◽

David J. Miller

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Rna Polymerase ◽

Intracellular Transport ◽

Protein A ◽

Specific Inhibitor ◽

Heat Shock Protein 90 ◽

Membrane Association ◽

Flock House Virus ◽

Intracellular Membrane

ABSTRACT The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

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Nodavirus-Induced Membrane Rearrangement in Replication Complex Assembly Requires Replicase Protein A, RNA Templates, and Polymerase Activity

Journal of Virology ◽

10.1128/jvi.01495-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12492-12503 ◽

Cited By ~ 34

Author(s):

Benjamin G. Kopek ◽

Erik W. Settles ◽

Paul D. Friesen ◽

Paul Ahlquist

Keyword(s):

Rna Viruses ◽

Protein A ◽

Protein S ◽

Rna Replication ◽

Polymerase Activity ◽

Complex Assembly ◽

Genomic Rnas ◽

Replication Complex ◽

Silencing Pathways ◽

Positive Strand Rna

ABSTRACT Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ∼50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only “zippered” together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.

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