Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication.Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses

A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery

A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells

The TPR Domain in the Host Cyp40-like Cyclophilin Binds to the Viral Replication Protein and Inhibits the Assembly of the Tombusviral Replicase

Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded proteins acting either as susceptibility or resistance factors. Previous genome-wide screens and global proteomics approaches with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of cyclophilins, which are a large family of host prolyl isomerases, in TBSV replication. In this paper, we identified those members of the large cyclophilin family that interacted with the viral replication proteins and inhibited TBSV replication. Further characterization of the most effective cyclophilin, the Cyp40-like Cpr7p, revealed that it strongly inhibits many steps during TBSV replication in a cell-free replication assay. These steps include viral RNA recruitment inhibited via binding of Cpr7p to the RNA-binding region of the viral replication protein; the assembly of the viral replicase complex and viral RNA synthesis. Since the TPR (tetratricopeptide repeats) domain, but not the catalytic domain of Cpr7p is needed for the inhibitory effect on TBSV replication, it seems that the chaperone activity of Cpr7p provides the negative regulatory function. We also show that three Cyp40-like proteins from plants can inhibit TBSV replication in vitro and Cpr7p is also effective against Nodamura virus, an insect pathogen. Overall, the current work revealed a role for Cyp40-like proteins and their TPR domains as regulators of RNA virus replication

Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus

Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family

Lasp-1 Regulates Podosome Function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins

A Discontinuous RNA Platform Mediates RNA Virus Replication: Building an Integrated Model for RNA–based Regulation of Viral Processes

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA–RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA–based interaction spanning ∼3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes

Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake

International audienceNogo recombinant proteins To express Amino-Nogo, the human Nogo-A cDNA for residues 1±1,040 was ligated into pcDNA3.1MycHis (Invitrogen, Burlingame, California) with an in-frame Myc-His tag. We transfected this plasmid into HEK293T cells and Amino-Nogo was puri®ed with a Ni 2+ resin 10. The human Nogo-66 sequence was ligated into pcAP-5 (ref. 10) in frame with the signal sequence, 6´His tag and placental AP coding region. This plasmid was transfected into HEK293 cells, and secreted AP±Nogo was puri®ed by Ni 2+ af®nity chromatography. GST±Nogo-66 has been described 1. Nogo-66 receptor binding assays and expression cloning To detect AP±Nogo binding, cultures were washed with Hanks balanced salt solution containing 20 mM sodium HEPES, pH 7.05, and 1 mg ml-1 bovine serum albumin (BSA) (HBH). The plates were then incubated with AP±Nogo in HBH for 2 h at 23 8C. We detected and quanti®ed bound AP±Nogo as for AP±Sema3A 11. For saturation analysis of AP±Nogo bound to COS-7 cells, bound AP±Nogo protein was eluted with 1% Triton X-100. After heat inactivation of endogenous AP, we measured AP±Nogo using p-nitrophenyl phosphate as substrate. For expression cloning of a Nogo-66 receptor, pools of 5,000 arrayed clones from a mouse adult-brain cDNA library (Origene Technologies, Rockville, Maryland) were transfected into COS-7 cells, and AP±Nogo binding was assessed. We isolated single NgR cDNA clones by sib selection and sequenced them. A Myc±NgR vector was created in pSecTag2-Hygro (Invitrogen) using the signal peptide of pSecTag2 fused to Myc and residues 27±473 of NgR. Human NgR cDNA was predicted from a human genomic cosmid sequence (AC007663). Oligonucleotide primers based on the predicted human cDNA ampli®ed the cDNA from a human adult-brain cDNA library (Origene Technologies). To assess binding of Myc±NgR to the cell membrane, particulate fractions were treated with or without 5 U PI-PLC (Sigma, St. Louis, MO) per mg of HEK293T cell protein for 1 h at 30 8C in HBH. After centrifugation at 100,000g for 1 h, we analysed equal proportions of the soluble and particulate fractions. To assess the physical interaction of NgR with Nogo-66, we incubated the PI-PLC extract (50 mg total protein) with 10 mg GST±Nogo-66 or buffer, or 10 mg GST for 1 h at 23 8C. We added glutathione-coupled agarose to bind GST and associated proteins. We analysed bound proteins by anti-Myc immunoblot. RNA analysis For northern blots, 1 mg poly(A) + RNA from each adult mouse tissue on a nylon membrane (Origene Technologies) was hybridized with a full-length 32 P-labelled probe 1,12. We used digoxegenin-labelled riboprobes (nucleotides 1±1,222) and adult mouse brain sections 1,12 for in situ hybridization. The sense probe produced no signal. Nogo-66 receptor antibodies A GST±NgR (residues 27±447) fusion protein was puri®ed from Escherichia coli and used to immunize rabbits. We diluted immune serum 3,000-fold for immunoblots and 1,000fold for immunohistology on tissue-culture samples that had been ®xed by formalin. Staining of tissue was totally abolished by addition of 5 mg ml-1 GST±NgR. Cell spreading, neurite outgrowth and viral infection To measure spreading rates, subcon¯uent NIH 3T3 ®broblasts or COS-7 cells were plated for 1 h in serum-containing medium before ®xation and staining with rhodaminephalloidin. Glass coverslips were precoated with 100 mg ml-1 poly-L-lysine, washed, and then 3 ml drops of PBS containing 15 pmol Amino-Nogo, 15 pmol GST±Nogo-66, 15 pmol poly-Asp (M r 35 K, Sigma), or no protein were spotted and dried. We added soluble Nogo protein preparations (100 nM) at the time of plating. Amino-Nogo was added alone or after a pre-incubation with a twofold molar excess of anti-Myc 9E10 antibody, or with a twofold excess of anti-Myc plus a twofold excess of puri®ed goat anti-mouse IgG. Chick E5 spinal cord, chick E7±E13 DRG, chick E7 retina and mouse P4 cerebellar neuron culture, growth-cone-collapse assays and neurite-outgrowth assays have been described 1,10±13. Here, outgrowth from dissociated neurons was assessed after 12±24 h. For the substrate-bound experiments, glass chamber slides were coated with 100 mg ml-1 poly-L-lysine, washed, and then 3 ml drops of PBS containing 15 pmol Amino-Nogo, 15 pmol GST±Nogo-66, 15 pmol poly-Asp, or no protein were spotted and dried. After three PBS washes, we coated slides with 10 mg ml-1 laminin. After aspiration of laminin, dissociated neurons were added. For the soluble Nogo experiments, slides were coated with poly-Llysine and laminin in the same fashion, and then 100 nM Amino-Nogo, 100 nM clustered Amino-Nogo, or 100 nM GST±Nogo-66 was added to the culture medium at the time of plating. After 1 d in vitro, some DRG explants were treated for 30 min with 1 unit ml-1 PI-PLC (Sigma) before the growth-cone-collapse assay. An HSV-Myc±NgR stock was prepared as described 10. We infected E7 retinal explants for 24 h. We stained some cultures infected with HSV-Myc±NgR with anti-Myc antibody to verify protein expression. Error bars re¯ect the s.e.m. from 4±8 determinations

Investigations on the Tobacco necrosis virus D p60 replicase protein

Copyright: © 2013 Fang, Coutts. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedTobacco necrosis virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerasePeer reviewe

Coordinated Function of Cellular DEAD-Box Helicases in Suppression of Viral RNA Recombination and Maintenance of Viral Genome Integrity

The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5' end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication