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George P Lomonossoff - One of the best experts on this subject based on the ideXlab platform.
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When plant virology met Agrobacterium: the rise of the deconstructed clones
Plant Biotechnology Journal, 2015Co-Authors: Hadrien Peyret, George P LomonossoffAbstract:In the early days of molecular farming, Agrobacterium‐mediated stable genetic transformation and the use of plant virus‐based vectors were considered separate and competing technologies with complementary strengths and weaknesses. The demonstration that ‘agroinfection’ was the most efficient way of delivering virus‐based vectors to their target plants blurred the distinction between the two technologies and permitted the development of ‘deconstructed’ vectors based on a number of plant viruses. The tobamoviruses, potexviruses, tobraviruses, geminiviruses and Comoviruses have all been shown to be particularly well suited to the development of such vectors in dicotyledonous plants, while the development of equivalent vectors for use in monocotyledonous plants has lagged behind. Deconstructed viral vectors have proved extremely effective at the rapid, high‐level production of a number of pharmaceutical proteins, some of which are currently undergoing clinical evaluation.
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structural fingerprinting subgrouping of Comoviruses by structural studies of red clover mottle virus to 2 4 a resolution and comparisons with other Comoviruses
Journal of Virology, 2000Co-Authors: Anthony J Clark, Zhongguo Chen, Michael Shanks, Ying Li, T Schmidt, P Oxelfelt, George P Lomonossoff, John E JohnsonAbstract:Red clover mottle virus (RCMV) is a member of the Comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other Comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other Comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of Comoviruses into CPMV-like and BPMV-like viruses.
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studies on hybrid Comoviruses reveal the importance of three dimensional structure for processing of the viral coat proteins and show that the specificity of cleavage is greater in trans than in cis
Virology, 1999Co-Authors: Anthony J Clark, Michael Shanks, J Wellink, Peter Bertens, George P LomonossoffAbstract:Abstract A series of cowpea mosaic virus (CPMV)-based hybrid comoviral RNA-2 molecules have been constructed. In these, the region encoding both the large (L) and small (S) viral coat proteins was replaced by the equivalent region from bean pod mottle virus (BPMV). The hybrid RNA-2 molecules were able to replicate in cowpea protoplasts in the presence of CPMV RNA-1. Though processing of the hybrid polyproteins by the CPMV-specific 24K proteinase at the site between the 58/48K and L proteins could readily be achieved, no processing at the site between the L and S coat proteins could be obtained even when the sequence of amino acids between the two coat proteins was made CPMV-like. As a result, none of the hybrids was able to form functional virus particles, and they could not infect cowpea plants. Comparison with the processing of the L–S site in cis in reticulocyte lysates demonstrated that the requirements for processing are more stringent in trans than in cis . The results suggest that the L–S cleavage site is defined by more than just a linear sequence of amino acids and probably involves interactions between the L–S loop and the β barrels of the viral coat proteins.
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the 24 kda proteinases of Comoviruses are virus specific in cis as well as in trans
Journal of General Virology, 1996Co-Authors: Michael Shanks, Johannes T Dessens, George P LomonossoffAbstract:To investigate the specificity of comoviral 24 kDa (‘24K’) proteinases, a full-length cDNA copy of red clover mottle virus (RCMV) RNA 1 has been cloned downstream of a T7 promoter. Translation in rabbit reticulocyte lysates of in vitro transcripts from this clone resulted in the synthesis of a 200K protein which was processed in a manner similar to that of the equivalent protein from cowpea mosaic virus (CPMV). Full-length cDNA clones of the RNA 1 molecules of RCMV and CPMV were used to create hybrid RNA 1 molecules. RNA transcribed in vitro from these hybrids was translated in vitro and the ability of the 24K proteinase from one comovirus to cleave the 32K/170K processing site from the other assessed. The results of the experiments show that the 24K proteinases are virus-specific in cis.
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Comoviruses and enteroviruses share a t cell epitope
Virology, 1992Co-Authors: M A Beck, John E Johnson, Steven Tracy, Beth Ann G Coller, Nora M Chapman, G Hufnagel, George P LomonossoffAbstract:Abstract An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant Comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro . However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the Comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the Comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.b
John E Johnson - One of the best experts on this subject based on the ideXlab platform.
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Structures of Picorna-Like Plant Viruses: Implications and Applications
Advances in Virus Research, 2020Co-Authors: John E JohnsonAbstract:Publisher Summary Picorna-like plant viruses that include Como and Nepo genera of the Comoviridae, share similarities in structure, genome organization, and replication strategy with mammalian picornaviruses. Structural studies of Comoviruses and a nepovirus contribute to the understanding of icosahedral virus structures, the development of novel methods for investigating macromolecular assemblies, and the infrastructure for virus-based bio- and nanotechnology. It is found that bean pod mottle virus (BPMV) shares similar overall genome organization, replication strategy, and capsid structure with the type member cowpea mosaic virus (CPMV). Structural studies of BPMV provided the first observation of a portion of a viral genome in association with its capsid, and the structural comparison of a nucleoprotein particle with an empty capsid that demonstrated ordering of elements of the capsid. Red clover mottle virus (RCMV) is another member of the comovirus family that was investigated biochemically and genetically and its structure was determined for comparative studies. There are significant structural differences between Comoviruses and some nepoviruses in that the capsids of Comoviruses are composed of two polypeptides whereas those of nepoviruses are composed of a single polypeptide. The structure of a nepovirus, tobacco ringspot virus (TRSV), was determined. Structural comparison showed that TRSV was likely to be an evolutionary intermediate of development of Comoviruses.
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structural fingerprinting subgrouping of Comoviruses by structural studies of red clover mottle virus to 2 4 a resolution and comparisons with other Comoviruses
Journal of Virology, 2000Co-Authors: Anthony J Clark, Zhongguo Chen, Michael Shanks, Ying Li, T Schmidt, P Oxelfelt, George P Lomonossoff, John E JohnsonAbstract:Red clover mottle virus (RCMV) is a member of the Comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other Comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other Comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of Comoviruses into CPMV-like and BPMV-like viruses.
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The refined crystal structure of cowpea mosaic virus at 2.8 A resolution.
Virology, 1999Co-Authors: Zhongguo Chen, T Schmidt, R. Usha, Cynthia V. Stauffacher, John E JohnsonAbstract:Abstract Comoviruses are a group of plant viruses in the picornavirus superfamily. The type member of Comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 A and the hexagonal space group P6 1 22, a = 451 A, c = 1038 A. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-A capsid was similar to the picornavirus capsid displaying a pseudo T = 3 ( P = 3) surface lattice. The three β-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the β-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 A resolution, despite the large unit cell. The large (∼200 A) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6 1 22 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell.
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The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily
Structure, 1998Co-Authors: Veda Chandrasekar, John E JohnsonAbstract:BACKGROUND: Tobacco ringspot virus (TRSV) is a member of the nepovirus genus of icosahedral RNA plant viruses that cause disease in fruit crops. Nepoviruses, Comoviruses and picornaviruses are classified in the picornavirus superfamily. Crystal structures of Comoviruses and picornaviruses and the molecular mass of the TRSV subunit (sufficient to accommodate three beta-barrel domains) suggested that nepoviruses may represent a link in the evolution of the picornavirus capsids from a T = 3 icosahedral virus. This evolutionary process is thought to involve triplication of the capsid protein gene, to encode a three-domain polyprotein, followed by development of cleavage sites in the interdomain linking regions. Structural studies on TRSV were initiated to determine if the TRSV subunit corresponds to the proposed uncleaved three-domain polyprotein. RESULTS: The 3.5 A resolution structure of TRSV shows that the capsid protein consists of three beta-barrel domains covalently linked by extended polypeptides. The order of connectivity of the domains in TRSV confirms the proposed connectivity for the precleaved comovirus and picornavirus capsid polyprotein. Structural differences between equivalent domains in TRSV and Comoviruses are confined to the external surface loops, interdomain connecting polypeptides and N termini. The three different domains within TRSV and Comoviruses are more closely related at the structural level than the three individual domains within picornaviruses. CONCLUSIONS: The structural results confirm the notion of divergent evolution of the capsid polyproteins of nepoviruses, Comoviruses and picornaviruses from a common ancestor. A number of residues were found to be conserved among various nepoviruses, some of which stabilize the quaternary structure of the three domains in the TRSV capsid protein subunit. Two conserved regions were identified on the external surface of TRSV, however, mutational studies will be needed to understand their functional significance. Nepoviruses transmitted by the same nematode species do not share regions with similar amino acid composition on the viral surface.
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Comoviruses and enteroviruses share a t cell epitope
Virology, 1992Co-Authors: M A Beck, John E Johnson, Steven Tracy, Beth Ann G Coller, Nora M Chapman, G Hufnagel, George P LomonossoffAbstract:Abstract An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant Comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro . However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the Comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the Comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.b
George Bruening - One of the best experts on this subject based on the ideXlab platform.
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Evidence for participation of RNA 1-encoded elicitor in Cowpea mosaic virus-mediated concurrent protection.
Virology, 2000Co-Authors: George Bruening, Jamal M. Buzayan, Carmen FerreiroAbstract:Abstract The cowpea ( Vigna unguiculata ) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus ; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non- Comoviruses : Cherry leafroll virus and Southern bean mosaic virus . Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus .
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Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.
Virology, 1992Co-Authors: Xiaojiang S Chen, George BrueningAbstract:Abstract Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5′-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5′-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24 K 87 K junction has a distinct as pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of as sequences from three Comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.
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nucleotide sequence and genetic map of cowpea severe mosaic virus rna 2 and comparisons with rna 2 of other Comoviruses
Virology, 1992Co-Authors: Xiaojiang S Chen, George BrueningAbstract:Abstract We report the nucleotide sequence of cowpea severe mosaic comovirus (CPSMV) genomic RNA 2. The molecule is composed of 3732 nucleotide (nt) residues, exclusive of the polyadenylate at the 3′ end. Only one of the six reading frame registers has a long open reading frame, from nt 255 to nt 3260 in the polarity of encapsidated RNA and corresponding to a polyprotein of 1002 amino acid residues (aa). As has been reported for other Comoviruses, a second in-frame AUG, at nt position 531, apparently also initiates translation, at least in vitro. Multiple alignments of the deduced CPSMV polyprotein as sequence with those of bean pod mottle comovirus (BPMV), cowpea mosaic como-virus (CPMV), and red clover mottle comovirus (RCMV) were consistent with a similar size for each of the three genes: the putative movement protein, beginning at the second in-frame AUG, the large coat protein (L), and the small coat protein. Identical nucleotide sequences in the terminal noncoding regions of RNA 2 of the four viruses are limited to 9 nt at the 5′ end and the 3′ polyadenylate. However, extensive similarities in sequence and potential structure were found. For all three genes and the 5' untranslated region, CPSMV and BPMV are more similar to each other than either is to CPMV or RCMV, the last two being similar to each other. Observed similarities predict that both cleavage sites in the CPSMV RNA 2 polyprotein are at glutamine-serine dipeptides. A sequence of 16 as at the amino terminus of L, determined by automated Edman degradation, matched a region of the deduced as sequence in the polyprotein and is consistent with cleavage at the predicted glutamine-serine dipeptide.
J. I. Cooper - One of the best experts on this subject based on the ideXlab platform.
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Complete sequences and phylogenetic analyses of a Singapore isolate of broad bean wilt fabavirus
Archives of Virology, 2001Co-Authors: J. I. Cooper, S. M. WongAbstract:The complete nucleotide (nt) sequence of a Singapore isolate of broad bean wilt fabavirus from Megakepasma erythrochlamys L., designated BBWV-ME , was determined. Its bipartite genome consisted of two positive-sense single-stranded ribonucleic acids (RNA). RNA1 (5951 nt in length) encoded a putative protease cofactor, nucleotide triphosphate (NTP)-binding domain (helicase), viral genome-linked protein (VPg), protease and RNA-dependent RNA polymerase (RdRp). RNA2 (3607 nt in length) encoded a putative movement protein (MP) and coat proteins (CP). Genome organization of BBWV-ME was similar to other viruses in the Comoviridae family. Phylogenetic analyses showed that fabaviruses were more closely related to the Comoviruses than the nepoviruses.
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Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus.
Journal of General Virology, 1994Co-Authors: S. Kreiah, G. Strunk, J. I. CooperAbstract:The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The N-terminal amino acid sequences of virion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97% identical to that of SLRSV-H. Except in the 3′ and 5′ terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the Comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.
R L S Forster - One of the best experts on this subject based on the ideXlab platform.
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nucleotide sequence of the coat protein genes of strawberry latent ringspot virus lack of homology to the nepoviruses and Comoviruses
Journal of General Virology, 1994Co-Authors: K R Everett, K S Milne, R L S ForsterAbstract:The sequence of the 3′-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3′ untranslated region of 552 nucleotides and a 3′-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M r of 43K is located 5′ of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and Comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and Comoviruses than has been considered previously.