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1.
J Gen Virol ; 102(12)2021 12.
Article in English | MEDLINE | ID: mdl-34914573

ABSTRACT

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.


Subject(s)
Bombyx/enzymology , Glutamate Dehydrogenase/metabolism , HSP70 Heat-Shock Proteins/metabolism , Peptidyl-Dipeptidase A/metabolism , Reoviridae/enzymology , Virus Attachment , Animals , Chromatography, Liquid , Immunoprecipitation , Reoviridae/physiology , Tandem Mass Spectrometry , Viral Structural Proteins/metabolism
3.
Viruses ; 9(4)2017 04 01.
Article in English | MEDLINE | ID: mdl-28368302

ABSTRACT

Dendrolimus punctatus cypovirus (DpCPV) is an important pathogen of D. punctatus, but little is known about the mechanisms of DpCPV infection. Here, we investigated the effects of VP3, VP4 and VP5 structural proteins on the viral invasion. Both the C-terminal of VP3 (methyltransferase (MTase) domain) and VP4 (A-spike) bound to Spodoptera exigua midgut brush border membrane vesicles (BBMVs) in a dose-dependent manner, and the binding was inhibited by purified DpCPV virions. Importantly, anti-MTase and anti-VP4 antibodies inhibited viral binding to S. exigua BBMVs. Using far-Western blots, a 65 kDa protein in Bombyx mori BBMVs, identified as alkaline phosphatase protein (BmALP) by mass spectrometry, specifically interacted with DpCPV MTase. The interaction between MTase and BmALP was verified by co-immunoprecipitation in vitro. Pretreatment of B. mori BBMVs with an anti-ALP antibody or incubation of DpCPV virions with prokaryotically expressed BmALP reduced viral attachment. Additionally, BmALP inhibited DpCPV infection in S. exigua larvae. Our data provide evidence that the MTase domain and A-spike function as viral attachment proteins during the DpCPV infection process, and ALP is the ligand that interacts with DpCPV via the MTase domain. These results augment our understanding of the mechanisms used by cypoviruses to enter their hosts.


Subject(s)
Alkaline Phosphatase/metabolism , Bombyx/enzymology , Methyltransferases/metabolism , Reoviridae/enzymology , Reoviridae/physiology , Viral Structural Proteins/metabolism , Virus Attachment , Animals , Immunoprecipitation , Methyltransferases/genetics , Protein Binding , Reoviridae/genetics , Spodoptera/virology , Viral Structural Proteins/genetics
4.
J Mol Biol ; 429(1): 79-87, 2017 01 06.
Article in English | MEDLINE | ID: mdl-27914893

ABSTRACT

Single-particle cryo-electron microscopy (cryo-EM) allows the high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5Å) cryo-EM structures reported to date were obtained by using 300kV transmission electron microscopes (TEMs). We report here the structures of a cypovirus capsid of 750-Å diameter at 3.3-Å resolution and of RNA-dependent RNA polymerase (RdRp) complexes within the capsid at 3.9-Å resolution using a 200-kV TEM. The newly resolved structure revealed conformational changes of two subdomains in the RdRp. These conformational changes, which were involved in RdRp's switch from non-transcribing to transcribing mode, suggest that the RdRp may facilitate the unwinding of genomic double-stranded RNA. The possibility of 3-Å resolution structural determinations for biological assemblies of relatively small sizes using cryo-EM at 200kV was discussed.


Subject(s)
Capsid/ultrastructure , Cryoelectron Microscopy , Macromolecular Substances/ultrastructure , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/ultrastructure , Reoviridae/ultrastructure , Models, Molecular , Protein Conformation , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , Reoviridae/enzymology , Reoviridae/metabolism
5.
Bing Du Xue Bao ; 32(5): 619-26, 2016 09.
Article in Chinese | MEDLINE | ID: mdl-30003768

ABSTRACT

In order to develop a novel effective biological insecticide for controlling oleander hawk moth, a new pathogen was isolated from naturally diseased Daphnis nerii. Based on scanning electron microscopy, full-length amplification of cDNAs (FLAC), and phylogenetic analysis of genome segments 2and 10,the virus was identified as a new type of cypovirus (Da phnis nerii cypovirus [DnCPV]). Electrophoresis analysis showed that DnCPV had a genome comprising 10double-stranded RNA (dsRNA) segments, ranging from 892 to 4160bp.Using FLAC, the cDNAs from the 10 dsRNA segments of the new CPV were cloned and genome segments 2and 10 were sequenced. Sequencing results showed that segment 2 encoded RNA-dependent-RNA-polymerases (RdRps) and segment 10 encoded polyhedrin. These two segments shared conserved terminal sequences of AGUCAAA and AGC at the 5'and 3'ends,respectively.These conserved terminal sequences were not consistent with any of the known CPV types.Phylogenetic analysis of the RdRp and polyhedrin indicated that this CPV was more closely related to CPV type 19 and type 5than other CPV types. Based on the unique conserved terminal sequences and the electrophoresis pattern of the new virus, we tentatively named it DnCPV Nanchang isolate: DnCPV-NC.


Subject(s)
Insect Viruses/isolation & purification , Moths/virology , Reoviridae/isolation & purification , Animals , Genome, Viral , Insect Viruses/classification , Insect Viruses/genetics , Phylogeny , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Reoviridae/classification , Reoviridae/enzymology , Reoviridae/genetics , Viral Proteins/genetics
6.
Nature ; 527(7579): 531-534, 2015 Nov 26.
Article in English | MEDLINE | ID: mdl-26503045

ABSTRACT

Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.


Subject(s)
Genome, Viral , Multienzyme Complexes/ultrastructure , RNA, Double-Stranded/ultrastructure , RNA, Viral/ultrastructure , RNA-Dependent RNA Polymerase/ultrastructure , Reoviridae/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Capsid Proteins/ultrastructure , Catalytic Domain , Cryoelectron Microscopy , Genome, Viral/genetics , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Nucleoside-Triphosphatase/metabolism , Nucleoside-Triphosphatase/ultrastructure , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA, Double-Stranded/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Messenger/ultrastructure , RNA, Viral/biosynthesis , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Reoviridae/enzymology , Reoviridae/genetics , Templates, Genetic , Transcription, Genetic
7.
Science ; 349(6254): 1347-50, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26383954

ABSTRACT

Double-stranded RNA (dsRNA) viruses possess a segmented dsRNA genome and a number of RNA-dependent RNA polymerases (RdRps) enclosed in a capsid. Until now, the precise structures of genomes and RdRps within the capsids have been unknown. Here we report the structures of RdRps and associated RNAs within nontranscribing and transcribing cypoviruses (NCPV and TCPV, respectively), using a combination of cryo-electron microscopy (cryo-EM) and a symmetry-mismatch reconstruction method. The RdRps and associated RNAs appear to exhibit a pseudo-D3 symmetric organization in both NCPV and TCPV. However, the molecular interactions between RdRps and the genomic RNA were found to differ in these states. Our work provides insight into the mechanisms of the replication and transcription in dsRNA viruses and paves a way for structural determination of lower-symmetry complexes enclosed in higher-symmetry structures.


Subject(s)
Capsid Proteins/ultrastructure , RNA, Double-Stranded/ultrastructure , RNA, Viral/ultrastructure , RNA-Dependent RNA Polymerase/ultrastructure , Reoviridae , Animals , Capsid/enzymology , Capsid/ultrastructure , Cryoelectron Microscopy , Genome, Viral , Humans , Protein Conformation , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Reoviridae/enzymology , Reoviridae/genetics , Reoviridae/ultrastructure , Transcription, Genetic , Virus Assembly
8.
J Mol Graph Model ; 61: 160-74, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26264734

ABSTRACT

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) contains 11 double stranded RNA genome segments and infects tasar silkworm A. mylitta. RNA-dependent RNA polymerase (RdRp) is reported as a key enzyme responsible for propagation of the virus in the host cell but its structure function relationship still remains elusive. Here a computational approach has been taken to compare sequence and secondary structure of AmCPV RdRp with other viral RdRps to identify consensus motifs. Then a reliable pairwise sequence alignment of AmCPV RdRp with its closest sequence structure homologue λ3 RdRp is done to predict three dimensional structure of AmCPV RdRp. After comparing with other structurally known viral RdRps, important sequence and/or structural features involved in substrate entry or binding, polymerase reaction and the product release events have been identified. A conserved RNA pentanucleotide (5'-AGAGC-3') at the 3'-end of virus genome is predicted as cis-acting signal for RNA synthesis and its docking and simulation study along with the model of AmCPV RdRp has allowed to predict mode of template binding by the viral polymerase. It is found that template RNA enters into the catalytic center through nine sequence-independent and two sequence-dependent interactions with the specific amino acid residues. However, number of sequence dependent interactions remains almost same during 10 nano-second simulation time while total number of interactions decreases. Further, docking of N(7)-methyl-GpppG (mRNA cap) on the model as well as prediction of RNA secondary structure has shown the template entry process in the active site. These findings have led to postulate the mechanism of RNA-dependent RNA polymerization process by AmCPV RdRp. To our knowledge, this is the first report to evaluate structure function relationship of a cypoviral RdRp.


Subject(s)
Dinucleoside Phosphates/chemistry , Genome, Viral , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/chemistry , Reoviridae/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Sequence Data , Moths/virology , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Reoviridae/enzymology , Sequence Alignment , Structural Homology, Protein , Substrate Specificity
9.
Elife ; 4: e07901, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26240998

ABSTRACT

mRNA transcription in dsRNA viruses is a highly regulated process but the mechanism of this regulation is not known. Here, by nucleoside triphosphatase (NTPase) assay and comparisons of six high-resolution (2.9-3.1 Å) cryo-electron microscopy structures of cytoplasmic polyhedrosis virus with bound ligands, we show that the large sub-domain of the guanylyltransferase (GTase) domain of the turret protein (TP) also has an ATP-binding site and is likely an ATPase. S-adenosyl-L-methionine (SAM) acts as a signal and binds the methylase-2 domain of TP to induce conformational change of the viral capsid, which in turn activates the putative ATPase. ATP binding/hydrolysis leads to an enlarged capsid for efficient mRNA synthesis, an open GTase domain for His217-mediated guanylyl transfer, and an open methylase-1 domain for SAM binding and methyl transfer. Taken together, our data support a role of the putative ATPase in mediating the activation of mRNA transcription and capping within the confines of the virus.


Subject(s)
Adenosine Triphosphatases/metabolism , RNA, Double-Stranded/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Reoviridae/enzymology , Reoviridae/genetics , Transcription, Genetic , Cryoelectron Microscopy , Reoviridae/ultrastructure , Viral Proteins/metabolism
10.
Gene ; 565(1): 56-61, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-25839934

ABSTRACT

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA.


Subject(s)
Bombyx/virology , RNA Interference , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Reoviridae/physiology , Animals , Gene Expression Profiling , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Larva/virology , RNA-Dependent RNA Polymerase/genetics , Reoviridae/enzymology , Virus Replication
11.
J Mol Biol ; 426(11): 2167-74, 2014 May 29.
Article in English | MEDLINE | ID: mdl-24690366

ABSTRACT

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2'-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-L-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.


Subject(s)
Methyltransferases/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Reoviridae/enzymology , Transcription, Genetic , Viral Regulatory and Accessory Proteins/chemistry , Catalytic Domain/genetics , Methyltransferases/genetics , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Secondary , Reoviridae/genetics , Viral Regulatory and Accessory Proteins/genetics
12.
Virol Sin ; 29(2): 86-93, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24643934

ABSTRACT

The double-shelled grass carp reovirus (GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene segment 2 (S2), is the putative RNA-dependent RNA polymerase (RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity (denoted as rVP2(390-900)) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2 (rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence (IF) assays, together with immunoblot (IB) analyses from both expressed cell extracts and purified Histagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg(2+), and was optimal at 28 °C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.


Subject(s)
RNA-Dependent RNA Polymerase/metabolism , Reoviridae/enzymology , Viral Structural Proteins/metabolism , Animals , Baculoviridae , Enzyme Activators/metabolism , Gene Expression , Genetic Vectors , Magnesium/metabolism , RNA-Dependent RNA Polymerase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reoviridae/genetics , Sf9 Cells , Spodoptera , Temperature , Viral Structural Proteins/genetics
13.
Curr Opin Struct Biol ; 19(6): 775-82, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19914820

ABSTRACT

RNA-dependent RNA polymerases (RdRps) of the segmented double-stranded (ds) RNA viruses of the Reoviridae family exhibit distinguishing structural elements, enabling the enzymes to function within the confines of a proteinaceous core particle. These globular, cage-like polymerases are traversed by four well-defined tunnels, which not only allow template RNAs, nucleotides, and divalent cations to access the interior catalytic site, but also provide two distinct exit conduits for RNA templates and products--one leading out of the core and the other back inside the core. Although Reoviridae RdRps are intrinsically capable of binding template, their catalytic activities are tightly regulated by interactions with core shell proteins. This intra-particle mechanism of RNA synthesis coordinates genome packaging with replication during the infectious cycle.


Subject(s)
RNA-Dependent RNA Polymerase/chemistry , Reoviridae/enzymology , Protein Structure, Tertiary , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism
14.
Virology ; 377(1): 7-18, 2008 Jul 20.
Article in English | MEDLINE | ID: mdl-18486179

ABSTRACT

The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.


Subject(s)
Adenosine Triphosphatases/genetics , Moths/virology , Reoviridae/enzymology , Reoviridae/genetics , Viral Structural Proteins/genetics , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Catalytic Domain , Cloning, Molecular , DNA, Viral/genetics , Genome, Viral , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism
15.
Arch Virol ; 152(7): 1341-51, 2007.
Article in English | MEDLINE | ID: mdl-17370108

ABSTRACT

Viruses of the species Mal de Río Cuarto virus (genus Fijivirus, family Reoviridae) cause significant economic losses in maize in Argentina. Genetic changes in the virus genome leading to better adaptation to diverse ecological conditions were postulated that would account for the increasing MRCV variability. The genomic differences between MRCV isolates from four ecologically different areas (Río Cuarto, RC; Pergamino, P; Jesús María, JM; and Tafí del Valle, TV) were studied. RT-PCR-amplified fragments comprising four genomic segments (Seg1, Seg7, Seg9 and Seg10) of MRCV isolates were compared by RFLPs and nucleotide sequences. The segments were chosen based on the proteins they encode: RNA-dependent-RNA polymerase, proteins putatively associated with tubular structures and viroplasm and the major outer capsid protein, respectively. Genetic comparison suggested that JM and TV isolates were genetically similar, but RC and P were different. Therefore, they were clustered in three genetic groups (JM = TV, RC and P). Together, nucleotide and amino acid sequence identities of the genomic segments were often above 96%. Seg1 was more variable (viral polymerase), whereas Seg7 (putative tubular structure) was the most conserved. Phylogeny analysis showed that MRCV isolates could be clustered in 'mountain area' and 'high production area' groups according to their geographical occurrence.


Subject(s)
Reoviridae/genetics , Amino Acid Sequence , Argentina , Base Sequence , DNA Primers/genetics , DNA, Viral/genetics , Ecosystem , Genetic Variation , Genome, Viral , Molecular Sequence Data , Phylogeny , Plant Diseases/virology , Polymorphism, Restriction Fragment Length , RNA-Dependent RNA Polymerase/genetics , Reoviridae/classification , Reoviridae/enzymology , Reoviridae/isolation & purification , Sequence Homology, Amino Acid , Zea mays/virology
16.
J Gen Virol ; 88(Pt 1): 342-350, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17170467

ABSTRACT

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1-S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1-S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [alpha-(32)P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133-667. Within this region, a sequence stretch (aa 170-250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of 'turreted' reoviruses, these results supported the a/vxxHx(8)Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.


Subject(s)
Genome, Viral , Nucleotidyltransferases/metabolism , Reoviridae/enzymology , Animals , Baculoviridae/genetics , Gene Expression , Genetic Vectors , Nucleotidyltransferases/genetics , RNA, Viral/analysis , RNA, Viral/metabolism , Reoviridae/genetics
17.
Arch Virol ; 149(4): 773-7, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15045563

ABSTRACT

The nucleotide sequences of genome segments 1 and 3 of Rosellinia anti-rot virus (RArV) from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix were determined. The complete nucleotide sequence of the genome segment 1 encoded a putative RNA-dependent RNA polymerase (RDRP). The deduced amino acid sequence of RDRP of RArV showed 29% identity with RDRPs of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, and identities of 23-21% with members of the genera Fijivirus and Cypovirus. Both RArV and the Coltivirus member might have originated from a common virus ancestor.


Subject(s)
Genome, Viral , Reoviridae/genetics , Ascomycota/virology , Molecular Sequence Data , RNA-Dependent RNA Polymerase/genetics , Reoviridae/classification , Reoviridae/enzymology , Sequence Homology, Nucleic Acid , Species Specificity
18.
Virology ; 316(2): 313-24, 2003 Nov 25.
Article in English | MEDLINE | ID: mdl-14644613

ABSTRACT

Grass carp reovirus, a segmented double-stranded RNA virus, is a member of the genus aquareovirus in the Reoviridae family. Grass carp reovirus VP1 was shown to be an mRNA guanylyltransferase. The enzyme demonstrated maximum activity

Subject(s)
Capsid Proteins/chemistry , Carps/virology , Nucleotidyltransferases/chemistry , Reoviridae/enzymology , Amino Acid Sequence , Animals , Capsid Proteins/metabolism , Chlorides/pharmacology , Guanosine Triphosphate/metabolism , Histidine , Hydrogen-Ion Concentration , Molecular Sequence Data , Nucleotidyltransferases/metabolism , Substrate Specificity , Sulfates/pharmacology
19.
Nat Struct Biol ; 10(12): 1011-8, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14608373

ABSTRACT

Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 A by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase lambda 3, were then fitted into the density map. Each copy of lambda 3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein lambda 1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of lambda 3 is bound per vertex. lambda 3 is furthermore oriented with its transcript exit channel facing a small channel through the lambda 1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein lambda 2.


Subject(s)
DNA-Directed RNA Polymerases/ultrastructure , RNA, Double-Stranded/ultrastructure , RNA-Dependent RNA Polymerase/ultrastructure , Reoviridae/enzymology , Reoviridae/ultrastructure , Viral Proteins/ultrastructure , Cryoelectron Microscopy , DNA-Directed RNA Polymerases/chemistry , Image Processing, Computer-Assisted , Nucleic Acid Conformation , Protein Conformation , RNA, Viral/ultrastructure , RNA-Dependent RNA Polymerase/chemistry , Sensitivity and Specificity , Viral Proteins/chemistry , Virion/enzymology , Virion/ultrastructure
20.
Res Microbiol ; 154(4): 231-6, 2003 May.
Article in English | MEDLINE | ID: mdl-12798226

ABSTRACT

The scarce characterisation of the viral world has hampered our efforts to appreciate the magnitude and diversity of the viral domain. It appears that almost every species can be infected by a number of viruses. As our knowledge of viruses increases, it appears that this myriad of viruses may be organised into a reasonably low number of viral lineages including members infecting hosts belonging to different domains of life. Viruses belonging to a lineage share a common innate "self" that refers to structural and assembly principles of the virion. This hypothesis has a few consequences. All viruses are old, maybe preceding cellular life, and virus origins are polyphyletic, as opposed to the idea of a monophyletic origin of cellular life.


Subject(s)
Archaeal Viruses , Bacteriophages , Biological Evolution , Phylogeny , Viruses , Adenoviridae/ultrastructure , Archaeal Viruses/chemistry , Archaeal Viruses/genetics , Archaeal Viruses/ultrastructure , Bacteriophage PRD1/ultrastructure , Bacteriophage phi 6/enzymology , Bacteriophage phi 6/ultrastructure , Bacteriophages/chemistry , Bacteriophages/genetics , Bacteriophages/ultrastructure , Capsid Proteins , Eukaryotic Cells/virology , Reoviridae/enzymology , Reoviridae/ultrastructure , Virus Assembly , Viruses/chemistry , Viruses/enzymology , Viruses/genetics , Viruses/ultrastructure
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