The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Paul Ahlquist - One of the best experts on this subject based on the ideXlab platform.
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An Amphipathic a-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function
2016Co-Authors: Ling Liu, Arturo Diaz, Johan Den A Boon, Xiaofeng Wang, William M Westler, Adam H Steinberg, Paul AhlquistAbstract:Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA Replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA Replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA Replication templates at these sites to establish active Replication complexes. During Replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic a-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA Replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and Replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA Replication complex assembly and show tha
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RESEARCH ARTICLE Host ESCRT Proteins Are Required for Bromovirus RNA Replication Compartment Assembly and Function
2016Co-Authors: Arturo Diaz, Jiantao Zhang, Abigail Ollwerther, Paul AhlquistAbstract:Positive-strand RNA viruses genome Replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA Replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA Replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling ma-chinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA Replication and the fre-quency of viral Replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA Replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA Replication> 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA Replication proteins accu-mulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT re-cruitment and spherule assembly depend on different sets of protein-protein interaction
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Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication
2014Co-Authors: Linhui Hao, Michael Newton, Paul AhlquistAbstract:Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA Replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV Replication. Candidates for host genes modulating FHV Replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 Replication. Overall, 65 deletion strains were confirmed to show significant changes in the Replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV Replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV Replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV Replication, three are localized to mitochondria, where FHV RNA Replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV Replication, and the remaining 46 are in the cytoplasm, with functions enriche
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intersection of the multivesicular body pathway and lipid homeostasis in RNA Replication by a positive strand RNA virus
Journal of Virology, 2011Co-Authors: Arturo Diaz, Brandi Gancarz, Johan Den A Boon, Paul Ahlquist, Xiaofeng WangAbstract:Like many positive-strand RNA viruses, brome mosaic virus (BMV) RNA Replication occurs in membrane-invaginated vesicular compartments. BMV RNA Replication compartments show parallels with membrane-enveloped, budding retrovirus virions, whose release depends on the cellular multivesicular body (MVB) sorting pathway. BMV RNA Replication compartments are not released from their parent membranes, but might depend on MVB functions for membrane invagination. Prior results show that BMV RNA Replication is severely inhibited by deletion of the crucial MVB gene DOA4 or BRO1. We report here that involvement of DOA4 and BRO1 in BMV RNA Replication is not dependent on the MVB pathway's membrane-shaping functions but rather is due to their roles in recycling ubiquitin from MVB cargos. We show that deleting DOA4 or BRO1 inhibits the ubiquitination- and proteasome-dependent activation of homologous transcription factors Mga2p and Spt23p, which regulate many lipid metabolism genes, including the fatty acid desaturase gene OLE1, which is essential for BMV RNA Replication. However, Mga2p processing and BMV RNA Replication are restored by supplementing free ubiquitin, which is depleted in doa4Δ and bro1Δ cells. The results identify Mga2p and Spt23p processing and lipid regulation as sensitive targets of ubiquitin depletion and correctly predict multiple effects of modulating additional host genes RFU1, UBP6, and UFD3. Our results also show that BMV RNA Replication depends on additional Mga2p-regulated genes likely involved in lipid metabolism beyond OLE1. Among other points, these findings show the potential for blocking viral RNA Replication by modulating lipid synthesis at multiple levels.
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membrane shaping host reticulon proteins play crucial roles in viral RNA Replication compartment formation and function
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Arturo Diaz, Xiaofeng Wang, Paul AhlquistAbstract:Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA Replication is inhibited 80–90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2apol polymerase. However, 1a failed to induce ER Replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional Replication vesicles but reduced their diameter by 30–50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA Replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2apol. Thus, RHPs interact with 1a, are incorporated into RNA Replication compartments, and are required for multiple 1a functions in Replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of Replication vesicles.
Xiaofeng Wang - One of the best experts on this subject based on the ideXlab platform.
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cowpea chlorotic mottle bromovirus Replication proteins support template selective RNA Replication in saccharomyces cerevisiae
PLOS ONE, 2018Co-Authors: Bryan S. Sibert, Amanda K. Navine, Janice G Pennington, Xiaofeng WangAbstract:Positive-strand RNA viruses generally assemble RNA Replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA Replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA Replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA Replication, we tested the ability of CCMV to similarly support spherule formation and RNA Replication in yeast. Paralleling BMV, we found that CCMV RNA Replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others' genomic RNA3 in plants, neither supported detectable CCMV RNA3 Replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited Replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the Replication complex. Overall, we show that many 1a and 2apol functions essential for Replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA Replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the Replication complex.
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An Amphipathic a-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function
2016Co-Authors: Ling Liu, Arturo Diaz, Johan Den A Boon, Xiaofeng Wang, William M Westler, Adam H Steinberg, Paul AhlquistAbstract:Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA Replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA Replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA Replication templates at these sites to establish active Replication complexes. During Replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic a-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA Replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and Replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA Replication complex assembly and show tha
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Host ESCRT proteins are required for bromovirus RNA Replication compartment assembly and function.
PLOS Pathogens, 2015Co-Authors: Arturo Diaz, Jiantao Zhang, Abigail Ollwerther, Xiaofeng WangAbstract:Positive-strand RNA viruses genome Replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA Replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA Replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA Replication and the frequency of viral Replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA Replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA Replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA Replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA Replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.
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BMV RNA Replication is inhibited in specific ESCRT deletion yeast strains.
2015Co-Authors: Arturo Diaz, Jiantao Zhang, Abigail Ollwerther, Xiaofeng WangAbstract:(A) BMV specific RNA-dependent RNA Replication and subgenomic mRNA synthesis is initiated from a cDNA derivative of RNA3. DNA-dependent transcription produces an initial (+)RNA transcript that serves as a template for a 1a- and 2apol-dependent RNA3 Replication and sgRNA4 synthesis via a (-) RNA intermediate. (B) Total RNA extracts were obtained from wt or ESCRT deletion strains expressing 1a, 2apol, and RNA3 and accumulation of negative-strand RNA3 and positive-strand subgenomic RNA4 was measured by Northern blotting. Equal loading was verified by probing for 18S ribosomal RNA. Values represent the mean of four independent experiments, with each condition tested in triplicate in each experiment. A representative blot is shown. All samples were compared to the wt control using a two tailed student t-test. * p
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intersection of the multivesicular body pathway and lipid homeostasis in RNA Replication by a positive strand RNA virus
Journal of Virology, 2011Co-Authors: Arturo Diaz, Brandi Gancarz, Johan Den A Boon, Paul Ahlquist, Xiaofeng WangAbstract:Like many positive-strand RNA viruses, brome mosaic virus (BMV) RNA Replication occurs in membrane-invaginated vesicular compartments. BMV RNA Replication compartments show parallels with membrane-enveloped, budding retrovirus virions, whose release depends on the cellular multivesicular body (MVB) sorting pathway. BMV RNA Replication compartments are not released from their parent membranes, but might depend on MVB functions for membrane invagination. Prior results show that BMV RNA Replication is severely inhibited by deletion of the crucial MVB gene DOA4 or BRO1. We report here that involvement of DOA4 and BRO1 in BMV RNA Replication is not dependent on the MVB pathway's membrane-shaping functions but rather is due to their roles in recycling ubiquitin from MVB cargos. We show that deleting DOA4 or BRO1 inhibits the ubiquitination- and proteasome-dependent activation of homologous transcription factors Mga2p and Spt23p, which regulate many lipid metabolism genes, including the fatty acid desaturase gene OLE1, which is essential for BMV RNA Replication. However, Mga2p processing and BMV RNA Replication are restored by supplementing free ubiquitin, which is depleted in doa4Δ and bro1Δ cells. The results identify Mga2p and Spt23p processing and lipid regulation as sensitive targets of ubiquitin depletion and correctly predict multiple effects of modulating additional host genes RFU1, UBP6, and UFD3. Our results also show that BMV RNA Replication depends on additional Mga2p-regulated genes likely involved in lipid metabolism beyond OLE1. Among other points, these findings show the potential for blocking viral RNA Replication by modulating lipid synthesis at multiple levels.
Ralf Bartenschlager - One of the best experts on this subject based on the ideXlab platform.
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hepatitis c virus ns2 3 processing is required for ns3 stability and viral RNA Replication
Journal of Biological Chemistry, 2005Co-Authors: Sarah Welbourn, Ralf Bartenschlager, Robin Green, Isabelle Gamache, Serge Dandache, V Lohmann, Karen Meerovitch, Arnim PauseAbstract:Abstract The hepatitis C virus NS2/3 protease is responsible for cleavage of the viral polyprotein between nonstructural proteins NS2 and NS3. We show here that mutation of three highly conserved residues in NS2 (His952, Glu972, and Cys993) abrogates NS2/3 protease activity and that introduction of any of these mutations into subgenomic NS2-5B replicons results in complete inactivation of NS2/3 processing and RNA Replication in both stable and transient Replication assays. The effect of uncleaved NS2 on the various activities of NS3 was therefore explored. Unprocessed NS2 had no significant effect on the in vitro ATPase and helicase activities of NS3, whereas immunoprecipitation experiments demonstrated a decreased affinity of NS4A for uncleaved NS2/3 as compared with NS3. This subsequently resulted in reduced kinetics in an in vitro NS3 protease assay with the unprocessed NS2/3 protein. Interestingly, NS3 was still capable of efficient processing of the polyprotein expressed from a subgenomic replicon in Huh-7 cells in the presence of uncleaved NS2. Notably, we show that fusion with NS2 leads to the rapid degradation of NS3, whose activity is essential for RNA Replication. Finally, we demonstrate that uncleaved NS2/3 degradation can be prevented by the addition of a proteasome inhibitor. We therefore propose that NS2/3 processing is a critical step in the viral life cycle and is required to permit the accumulation of sufficient NS3 for RNA Replication to occur. The regulation of NS2/3 cleavage could constitute a novel mechanism of switching between viral RNA Replication and other processes of the hepatitis C virus life cycle.
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mutational analysis of hepatitis c virus nonstructural protein 5a potential role of differential phosphorylation in RNA Replication and identification of a genetically flexible domain
Journal of Virology, 2005Co-Authors: Nicole Appel, Thomas Pietschmann, Ralf BartenschlagerAbstract:Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA Replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA Replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA Replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the Replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA Replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA Replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on Replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.
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kissing loop interaction in the 3 end of the hepatitis c virus genome essential for RNA Replication
Journal of Virology, 2005Co-Authors: Peter Friebe, Julien Boudet, Jeanpierre Simorre, Ralf BartenschlagerAbstract:The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for Replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for Replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA Replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA Replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA Replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for Replication.
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viral and cellular determinants of hepatitis c virus RNA Replication in cell culture
Journal of Virology, 2003Co-Authors: Volker Lohmann, Sandra Hoffmann, Ulrike Herian, Francois Penin, Ralf BartenschlagerAbstract:Studies on the Replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA Replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA Replication. Mutations enhancing Replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on Replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on Replication but that enhanced Replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in Replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA Replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in inteRNAl ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that Replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV Replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.
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sequences in the 5 nontranslated region of hepatitis c virus required for RNA Replication
Journal of Virology, 2001Co-Authors: Peter Friebe, Volker Lohmann, Nicole Krieger, Ralf BartenschlagerAbstract:Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and Replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an inteRNAl ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA Replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA Replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA Replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA Replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA Replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV Replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for Replication of this virus.
Bert L. Semler - One of the best experts on this subject based on the ideXlab platform.
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functional consequences of RNA 5 terminal deletions on coxsackievirus b3 RNA Replication and ribonucleoprotein complex formation
Journal of Virology, 2017Co-Authors: Nicolas Leveque, Magali Garcia, Alexis Bouin, Joseph H C Nguyen, Genevieve P Tran, Laurent Andreoletti, Bert L. SemlerAbstract:Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5'-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5' stem-loop I, which is an RNA secondary structure required for viral RNA Replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5' deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5' deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5' stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA Replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5' end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA Replication observed for the 5'-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA Replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5' end of the viral genome involving an RNA secondary structure required for RNA Replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA Replication. In this report, we demonstrate that transfected RNAs harboring 5'-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral Replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.
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viral subversion of host functions for picoRNAvirus translation and RNA Replication
Future Virology, 2012Co-Authors: Amanda J Chase, Bert L. SemlerAbstract:PicoRNAvirus infections lead to symptoms that can have serious health and economic implications. The viruses in this family (PicoRNAviridae) have a small genomic RNA and must rely on host proteins for efficient viral gene expression and RNA Replication. To ensure their effectiveness as pathogens, picoRNAviruses have evolved to utilize and/or alter host proteins for the benefit of the virus life cycle. This review discusses the host proteins that are subverted during infection to aid in virus Replication. It will also describe proteins and functions that are altered during infection for the benefit of the virus.
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differential rescue of poliovirus RNA Replication functions by genetically modified RNA polymerase precursors
Journal of Virology, 2004Co-Authors: Christopher T Cornell, Jo Ellen Brunner, Bert L. SemlerAbstract: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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distinct poly rc binding protein kh domain determinants for poliovirus translation initiation and viral RNA Replication
Journal of Virology, 2002Co-Authors: Brandon L Walter, Todd B Parsley, Ellie Ehrenfeld, Bert L. SemlerAbstract:The limited coding capacity of picoRNAvirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picoRNAvirus RNAs containing type I inteRNAl ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus Replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA Replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA Replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA Replication. These predictions were confirmed in functional assays that allow separation of RNA Replication activities from translation. Our data have implications for differential picoRNAvirus template utilization during viral translation and RNA Replication and suggest that specific PCBP2 domains may have distinct roles in these activities.
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rescue of defective poliovirus RNA Replication by 3ab containing precursor polyproteins
Journal of Virology, 1998Co-Authors: Jonathan S Towner, Melissa M Mazanet, Bert L. SemlerAbstract:Poliovirus (PV), the prototypic picoRNAvirus, replicates its genomic RNA via membranous Replication complexes within the cytoplasm of an infected cell (11, 12). These complexes appear as rosette-like structures (8) and are thought to provide an environment for increased local concentrations of poliovirus proteins, presumably limiting diffusion within the Replication complex (22). Highly specific interactions between cellular and viral proteins associated with virion RNA (vRNA) and cellular membranes result in the formation of PV Replication complexes. These interactions include tight membrane-protein associations by PV proteins 3AB (17, 40, 45, 47, 52) and 2C (15, 20, 48) combined with specific RNA-protein interactions between the PV 5′ noncoding region (5′NCR) and the viral polypeptide 3CD (2, 3). In addition, the presence of the 5′ terminal ∼100 nucleotides (nt) of PV RNA mediates an in vitro interaction between 3CD and the cellular protein poly(rC) binding protein 2 (21, 36). The viral polypeptide 3AB (24, 54) and the cellular protein EF-1α (24) also appear to complex with 3CD and the 5′ end of the PV genome. Interactions between the viral proteins 3D and 3AB have been documented (25, 29, 37), as have RNA-protein interactions involving the 3′ ends of positive- and negative-strand picoRNAvirus RNAs (4, 39, 50). While many of these studies have focused on individual molecular interactions, little is known about the viral polyprotein subunits necessary for the initial assembly of the vRNA Replication complex. For PV, like all picoRNAviruses, mature gene products are specifically processed from viral precursor polyproteins (42). The efficient and highly regulated protein processing cascade produces cleavage products with functions distinct from those of their precursor proteins. Due to the short half-lives of large precursor proteins, it has been difficult to determine the composition of assembly intermediates. An additional control of RNA Replication relies on the translation of PV RNA; that is, translation of a particular genome is a prerequisite for that genome to be competent for Replication due to the requirement for either a cis-acting viral protein(s) or ribosomal passage over the RNA template (34). A relationship between ribosomal passage and RNA Replication stems from the observation that all naturally occurring defective interfering particles contain deletions in the capsid region (P1 [Fig. 1]) that maintain the reading frame (28). The requirement for translation prior to RNA Replication was later confirmed by using genetically engineered replicons in which Replication occurred only if deletions were in frame and the P2-P3 region was left intact (except for the N terminus of viral protein 2A) (16). The apparent cis dominance of translation over Replication does not in theory preclude the utilization of some of the viral nonstructural proteins in trans. For example, passage of the ribosome could transiently expose a cis-acting Replication determinant present on the RNA template, while the active Replication proteins in the complex could consist of mixed viral proteins, some of which were synthesized from other viral mRNAs. FIG. 1 Schematic representation of full-length and subgenomic PV RNAs. (A) Schematic representation of the PV genomic structure and viral protein organization where the viral structural proteins are derived from the P1 portion of the genome and the nonstructural ... Complementation of site-specific lesions in trans has been assayed by providing the wild-type gene product(s) through a helper virus (6, 7, 14, 18, 22, 26, 32, 49) and through novel approaches using dicistronic RNAs or amber-suppressing cell lines (13, 34). The collective results of such studies indicate that each viral protein likely has multiple functions, some of which can be complemented in trans and some of which cannot, depending on whether a mutation exerts its effect at the level of the precursor protein or at the level of the mature cleavage product. Previous experimental approaches often utilized a helper virus or helper RNA to provide the complementing gene products in infected or transfected cells. vRNAs produced in cells from such approaches most likely generate individual RNA Replication complexes that are physically separated from each other in the cytoplasm. By the time viral proteins are generated to levels sufficient for diffusion and effective complementation, polypeptides in the Replication complexes may be processed or assembled such that they are no longer competent for subunit exchange. In this study, we attempted to circumvent the problem of inaccessibility to the PV Replication complex by using a HeLa cell-free Replication assay to produce potential complementing viral proteins during the initial stages of complex formation. We addressed four questions. (i) What polyprotein subunit delivers the RNA Replication function of viral protein 3AB during assembly of the Replication complex? (ii) Can this 3AB-containing assembly intermediate by provided in trans? (iii) Does this assembly subunit need to be proteolytically active? (iv) Does 3AB need to be physically linked to the active viral RNA polymerase during complex assembly? Our results show that a 3AB mutation causing a severe RNA Replication defect can be efficiently complemented in trans by providing the large replicase precursor (P3) but not the mature 3AB polypeptide. Furthermore, the rescuing trans-P3 protein must contain an active 3C proteolytic domain but not an active polymerase domain. Possible mechanisms for complementation are discussed.
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brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting RNA Replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Tokiko Watanabe, Waiming Lee, Michael P Schwartz, Michael Janda, Paul AhlquistAbstract:Positive-strand RNA virus RNA Replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA Replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a(pol) has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a(pol), and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA Replication compartments. 1a also recruits BMV Replication templates such as genomic RNA3. In the absence of 2a(pol), 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a(pol). Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA Replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed Replication compartment is discussed.
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brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting RNA Replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Tokiko Watanabe, Waiming Lee, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic Replication mechanisms. For example, genome Replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral Replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus Replication, the viral genomic RNAs first serve as templates for translating these Replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated Replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA Replication. This genomic RNA transition from translation to RNA Replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and Replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for Replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode Replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA Replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA Replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA Replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA Replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA Replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-susceptible state in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This state appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant state, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled Replication complex. When preformed RNA Replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA Replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA Replication. Most Replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant state induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into Replication complexes.
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mutation of host δ9 fatty acid desaturase inhibits brome mosaic virus RNA Replication between template recognition and RNA synthesis
Journal of Virology, 2001Co-Authors: Paul Ahlquist, Masayuki Ishikawa, Waiming LeeAbstract:All positive-strand RNA viruses assemble their RNA Replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae . BMV encodes RNA Replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA Replication complexes on the perinuclear ER. We show that BMV RNA Replication is severely inhibited by a mutation in OLE1 , an essential yeast chromosomal gene encoding Δ9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA Replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA Replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA Replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA Replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.