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C M Fauquet

Publications and source records attributed to C M Fauquet.

At least 37 records · Page 2Linked to original sources
PubMed2000

Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon.

Stem cuttings were collected in Cameroon from cassava plants displaying cassava mosaic disease (CMD) symptoms. The nature of the viruses present was determined by using the PCR with primers specific for the coat protein (CP) genes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). All samples were infected by ACMV and eight of the 50 samples were infected by both ACMV and an EACMV-like virus. The complete nucleotide sequences of DNA-A and -B of representative ACMV and EACMV-like viruses were determined. The DNA-A component of the EACMV-like virus contained evidence of recombination in the AC2-AC3 region and DNA-B also contained evidence of recombination in BC1. However, both components retained gene arrangements typical of bipartite begomoviruses. When Nicotiana benthamiana plants were doubly inoculated with these Cameroon isolates of ACMV and EACMV (ACMV/CM, EACMV/CM) by using sap from cassava plants or infectious clones, the symptoms were more severe than for plants inoculated with either virus alone. Southern blot analysis of viral DNAs from infected plants showed that there were significantly higher levels of accumulation of both ACMV/CM components and, to a lesser extent, of EACMV/CM components in mixed-infected plants than in singly infected plants. These results strongly suggest the occurrence of a synergistic interaction between the two viruses.

Base Sequence↗
PubMed1999

Possible emergence of new geminiviruses by frequent recombination.

Although exchange of genetic information by recombination plays a role in the evolution of viruses, the extent to which it generates diversity is not clear. We analyzed genomes of geminiviruses for recombination using a new statistical procedure developed to detect gene conversions. Geminiviruses (family, Geminiviridae) are a group of plant viruses characterized by a genome of circular single-stranded DNA (approximately 2700 nucleotides in length) encapsidated in twinned quasi-isometric particles. Complete nucleotide sequences of geminiviruses were aligned, and recombination events were detected by searching pairs of viruses for sequences that are significantly more similar than expected based on random distribution of polymorphic sites. The analyses revealed that recombination is very frequent and occurs between species and within and across genera. Tests identified 420 statistically significant recombinant fragments distributed across the genome. The results suggest that recombination is a significant contributor to geminivirus evolution. The high rate of recombination may be contributing to the recent emergence of new geminivirus diseases.

Evolution, Molecular↗
PubMed1999

Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.

Animals↗
PubMed1999

Molecular characterization of a plant mitochondrial chaperone GrpE.

Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE).

Amino Acid Sequence↗
PubMed1999

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement.

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.

Bacteriophage M13↗
PubMed1999

Identification of replication specificity determinants in two strains of tomato leaf curl virus from New Delhi.

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.

Asparagine↗
PubMed1998

Expression and inheritance of multiple transgenes in rice plants.

The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.

Acetyltransferases↗
PubMed1996

The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus.

We analyzed various mutants of tomato leaf curl virus-India to investigate the role of ORFs AV3, AV2, and coat protein (CP) in viral replication, movement, and symptom development. The results of these studies indicate that ORF AV3 does not encode a protein. Plants inoculated with infectious DNA which contained deletions in AV2 developed very mild symptoms and accumulated only low levels of both single-stranded (ss) and double-stranded (ds) viral DNA, whereas inoculated protoplasts accumulated both ss and dsDNA to wild-type levels, showing that AV2 is required for efficient viral movement. However, both plants and protoplasts inoculated with substitution, frameshift, and other similar mutations in AV2 accumulated low levels of viral DNA. The low levels of accumulation of DNA of these mutants were apparently not due to a defect in AV2 synthesis. Mutations in the CP caused a marked decrease in ssDNA accumulation in plants and protoplasts while increasing dsDNA accumulation in protoplasts. Mutations in both AV2 and CP behaved like AV2 mutants in plants and like CP mutants in protoplasts. The results demonstrated that multiple functions provided by AV2, BV1, BC1 are essential for viral movement, and that changes in A-component virion-sense mRNA structure or translation affect viral replication.

Amino Acid Sequence↗
PubMed1996

Mass spectrometry and viral analysis.

BACKGROUND: Electrospray ionization (ESI) mass spectrometry is a powerful new approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry; it is not clear, however, whether the native conformation of the biomolecules is maintained throughout the ionization and analysis process. We set out to address this question using live viruses. RESULTS: Viral ions have been generated in the gas phase using electrospray ionization mass spectrometry. These ions have been collected, following ion filtering through the mass analyzer, and then analyzed by transmission electron microscopy. Transmission electron microscopy revealed that rice yellow mottle virus and tobacco mosaic virus retained their respective spherical and rod-like ultrastructure. The viability of the isolated tobacco mosaic virus was confirmed by inoculation and infection of tobacco plants. CONCLUSIONS: These results demonstrate the utility of electrospray for supramolecular complexes with molecular weights of over 40 million Da and offer conclusive evidence that native biomolecular structures can be conserved through the electrospray process.

Mass Spectrometry↗
PubMed1996

Characterization of cassava common mosaic virus and a defective RNA species.

The genome of cassava common mosaic potexvirus (CsCMV) has been sequenced from cDNA clones and consists of 6376 nucleotides (nt). A 76 nt untranslated region (UTR) at the 5' terminus was followed by ORF1 which potentially encodes a protein of 1449 amino acids (aa). ORFs 2, 3, and 4 were predicted to encode proteins of 231, 112 and 97 aa, respectively. ORF5 potentially encodes a 229 aa protein of 25 kDa that is similar to the coat proteins of other potexviruses. The 3'-terminal UTR of 114 nt was followed by a poly(A) tail. The genomic organization of the CsCMV genome is similar to that of other potexviruses. A cDNA clone that was apparently obtained from a defective RNA species contained both the 5' and 3' UTRs and an ORF that potentially encodes the first 263 aa of ORF1 and the last 33 aa of the coat protein. Defective RNA species were found both in purified preparations of the virus and in total nucleic acid isolated from CsCMV-infected plants.

Amino Acid Sequence↗