scholarly journals Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication†

Biochemistry ◽  
2007 ◽  
Vol 46 (17) ◽  
pp. 5185-5199 ◽  
Author(s):  
Kentaro Fujita ◽  
Shyam S. Krishnakumar ◽  
David Franco ◽  
Aniko V. Paul ◽  
Erwin London ◽  
...  
Keyword(s):  
Rna Replication ◽  
Membrane Association ◽  
Functional Effect ◽  
Membrane Topography ◽  
Anchor Sequence ◽  
Hydrophobic Anchor

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

2004 ◽  
Vol 78 (20) ◽  
pp. 11393-11400 ◽  
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.

scholarly journals Modification of Cellular Autophagy Protein LC3 by Poliovirus

2007 ◽  
Vol 81 (22) ◽  
pp. 12543-12553 ◽  
Author(s):  
Matthew P. Taylor ◽  
Karla Kirkegaard
Keyword(s):  
Biochemical Marker ◽  
Protein A ◽  
Rna Replication ◽  
Viral Proteins ◽  
Innate Immune ◽  
Host Protein ◽  
Membrane Association ◽  
Poliovirus Infection ◽  
Cellular Autophagy ◽  
Distinct Aspect

ABSTRACT Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.

scholarly journals Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal

2001 ◽  
Vol 75 (7) ◽  
pp. 3207-3219 ◽  
Author(s):  
Jianbo Chen ◽  
Amine Noueiry ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Protein ◽  
Membrane Association ◽  
Stem Loop ◽  
Induced Membrane ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

scholarly journals Anchor sequence-dependent endogenous processing of human immunodeficiency virus 1 envelope glycoprotein gp160 for CD4+ T cell recognition.

1990 ◽  
Vol 171 (3) ◽  
pp. 875-887 ◽  
Author(s):  
M Polydefkis ◽  
S Koenig ◽  
C Flexner ◽  
E Obah ◽  
K Gebo ◽  
...  
Keyword(s):  
T Cell ◽  
Envelope Glycoprotein ◽  
Recombinant Vaccinia Virus ◽  
Cd4 T Cell ◽  
Cellular Transport ◽  
Cell Clones ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Anchor Sequence ◽  
Hydrophobic Anchor

Human CD4+ T cell clones and cell lines were shown to lyse recombinant vaccinia virus-infected cells that synthesize the HIV-1 envelope glycoprotein gp160. The processing of endogenously synthesized gp160 for recognition by CD4+ T cells required that the protein, after synthesis on the rough endoplasmic reticulum and during subsequent cellular transport, remain attached to the luminal/extracellular membrane face by a hydrophobic anchor sequence.

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

2002 ◽  
Vol 76 (19) ◽  
pp. 9856-9867 ◽  
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.

scholarly journals Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication

2004 ◽  
Vol 78 (23) ◽  
pp. 13278-13284 ◽  
Author(s):  
Darius Moradpour ◽  
Volker Brass ◽  
Elke Bieck ◽  
Peter Friebe ◽  
Rainer Gosert ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Insertion Sequence ◽  
Rna Replication ◽  
Hcv Rna ◽  
Membrane Association ◽  
Rna Dependent Rna Polymerase ◽  
Terminal Domain

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.

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

2009 ◽  
Vol 83 (9) ◽  
pp. 4498-4507 ◽  
Author(s):  
Kenneth A. Stapleford ◽  
Doron Rapaport ◽  
David J. Miller
Keyword(s):  
Rna Polymerase ◽  
Protein A ◽  
Rna Replication ◽  
Mitochondrial Outer Membrane ◽  
Membrane Association ◽  
Flock House Virus ◽  
Replication Complex ◽  
Anionic Phospholipids ◽  
Positive Strand Rna

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.

scholarly journals RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins

2015 ◽  
Vol 90 (3) ◽  
pp. 1687-1692 ◽  
Author(s):  
Katri Kallio ◽  
Kirsi Hellström ◽  
Eija Jokitalo ◽  
Tero Ahola
Keyword(s):  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Rna Replication ◽  
Membrane Association ◽  
Nonstructural Proteins ◽  
Membrane Modification ◽  
Negative Strand ◽  
Replication System ◽  
Replication Sites ◽  
Rna Capping

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virustrans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

scholarly journals Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA

2007 ◽  
Vol 81 (9) ◽  
pp. 4633-4644 ◽  
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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