scholarly journals The Heat Shock Protein 70 Cochaperone YDJ1 Is Required for Efficient Membrane-Specific Flock House Virus RNA Replication Complex Assembly and Function in Saccharomyces cerevisiae

2007 ◽  
Vol 82 (4) ◽  
pp. 2004-2012 ◽  
Author(s):  
Spencer A. Weeks ◽  
David J. Miller
Keyword(s):  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Complex Activity ◽  
Complex Assembly ◽  
Replication Complex ◽  
Hsp90 Chaperone ◽  
Chaperone Complex ◽  
And Function

ABSTRACT The assembly of RNA replication complexes on intracellular membranes is an essential step in the life cycle of positive-sense RNA viruses. We have previously shown that Hsp90 chaperone complex activity is essential for efficient Flock House virus (FHV) RNA replication in Drosophila melanogaster S2 cells. To further explore the role of cellular chaperones in viral RNA replication, we used both pharmacologic and genetic approaches to examine the role of the Hsp90 and Hsp70 chaperone systems in FHV RNA replication complex assembly and function in Saccharomyces cerevisiae. In contrast to results with insect cells, yeast deficient in Hsp90 chaperone complex activity showed no significant decrease in FHV RNA replication. However, yeast with a deletion of the Hsp70 cochaperone YDJ1 showed a dramatic reduction in FHV RNA replication that was due in part to reduced viral RNA polymerase accumulation. Furthermore, the absence of YDJ1 did not reduce FHV RNA replication when the viral RNA polymerase and replication complexes were retargeted from the mitochondria to the endoplasmic reticulum. These results identify YDJ1 as an essential membrane-specific host factor for FHV RNA replication complex assembly and function in S. cerevisiae and are consistent with known differences in the role of distinct chaperone complexes in organelle-specific protein targeting between yeast and higher eukaryotes.

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

2005 ◽  
Vol 79 (11) ◽  
pp. 6827-6837 ◽  
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.

scholarly journals An Amphipathic α-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function

2009 ◽  
Vol 5 (3) ◽  
pp. e1000351 ◽  
Author(s):  
Ling Liu ◽  
William M. Westler ◽  
Johan A. den Boon ◽  
Xiaofeng Wang ◽  
Arturo Diaz ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Multiple Roles ◽  
Virus Protein ◽  
Complex Assembly ◽  
Replication Complex ◽  
Α Helix ◽  
And Function

scholarly journals BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

2016 ◽  
Vol 90 (15) ◽  
pp. 6864-6883 ◽  
Author(s):  
Morgan R. Herod ◽  
Cristina Ferrer-Orta ◽  
Eleni-Anna Loundras ◽  
Joseph C. Ward ◽  
Nuria Verdaguer ◽  
...  
Keyword(s):  
Disease Virus ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Rna Replication ◽  
Mouth Disease ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Complex ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

Role of Cellular Structures in Viral RNA Replication

2014 ◽  
pp. 247-253 ◽  
Author(s):  
Denise Egger ◽  
Rainer Gosert ◽  
Kurt Bienz
Keyword(s):  
Rna Replication ◽  
Viral Rna ◽  
Cellular Structures

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

2009 ◽  
Vol 83 (7) ◽  
pp. 2976-2988 ◽  
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.

scholarly journals Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion

2015 ◽  
Vol 89 (22) ◽  
pp. 11233-11244 ◽  
Author(s):  
Kenneth A. Stapleford ◽  
Kathryn Rozen-Gagnon ◽  
Pratyush Kumar Das ◽  
Sirle Saul ◽  
Enzo Z. Poirier ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Nucleoside Analogs ◽  
Viral Rna ◽  
High Fidelity ◽  
Viral Polymerase ◽  
Replication Fidelity ◽  
Resistant Variant ◽  
Replication Complex ◽  
Viral Replicase

ABSTRACTTo date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.IMPORTANCEPrevious studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.

Sign in / Sign up

Export Citation Format

Share Document