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M N Pearson

Publications and source records attributed to M N Pearson.

At least 19 recordsLinked to original sources
PubMed2006

Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect vanilla.

Sequence was determined for the coat protein (CP) gene and 3' non-translated region (3'NTR) of two vanilla mosaic virus (VanMV) isolates from Vanilla tahitensis, respectively from the Cook Islands (VanMV-CI) and French Polynesia (VanMV-FP). Both viruses displayed distinctive features in the N-terminal region of their CPs; for VanMV-CI, a 16-amino-acid deletion including the aphid transmission-related DAG motif, and for VanMV-FP, a stretch of GTN repeats that putatively belongs to the class of natively unfolded proteins. VanMV-FP CP also has a novel DVG motif in place of the DAG motif, and an uncommon Q//V protease cleavage site. The sequences were compared to a range of Dasheen mosaic virus (DsMV) strains and to potyviruses infecting orchids. Identity was low to DsMV strains across the entire CP coding region and across the 3'NTR, but high across the CP core and the CI-6K2-NIa region. In accordance with current ICTV criteria for species demarcation within the family Potyviridae, VanMV-CI and VanMV-FP are strains of DsMV that exclusively infect vanilla.

3' Untranslated Regions↗
PubMed2005

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant 'potex-like' viruses.

This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3' terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant 'potex-like' viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant 'potex-like' coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant 'potex-like' virus group, distinct from BVF.

Amino Acid Sequence↗
PubMed2001

Characterization of a baculovirus-encoded protein that is associated with infected-cell membranes and budded virions.

Two types of envelope fusion proteins have been identified in lepidopteran baculoviruses. GP64 is found in Autographa californica multinucleocapsid nucleopolyhedrovirus, Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (OpMNPV), and other relatively closely related viruses, while Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), which lacks GP64, utilizes LD130 as its envelope fusion protein. Homologs of ld130 have since been found not only in all the sequenced gp64-minus virus genomes, but also in the genomes of gp64-containing viruses. In addition, they are evolutionarily related to the envelope proteins of certain insect retroviruses. In this report, the characterization of a LD130 homolog (OP21) from OpMNPV, which also contains gp64, is described. Western blot analysis of extracts of OpMNPV-infected Lymantria dispar cells, using antibodies generated against OP21, identified an infected cell-specific doublet of 85 and 89 kDa. These bands were first observed at about 6 h p.i. and were present at all later time points. Such analyses also demonstrated that OP21 was associated with budded virions. Tunicamycin treatment of OpMNPV-infected cells indicated that OP21 is N-glycosylated. Studies employing NP-40 to remove the envelope from budded virions indicated that the majority of OP21 remained associated with the nucleocapsid fraction, whereas all GP64 was removed. Confocal immunofluorescence microscopy showed that OP21 and GP64 have a similar pattern of distribution on the membrane of cells infected with OpMNPV. Immunoelectron microscopy of budded virions also showed similar patterns of localization for both OP21 and GP64.

Amino Acid Sequence↗
PubMed2000

Identification of the lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae.

The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.

Animals↗
PubMed1999

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar.

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

Amino Acid Sequence↗
PubMed1998

Characterization of a baculovirus-encoded ATP-dependent DNA ligase.

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.

Amino Acid Sequence↗
PubMed1997

Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of the L dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer did not show increased activity when cotransfected with LdMNPV ie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system. LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of a second putative origin of DNA replication in a baculovirus of Orgyia pseudotsugata.

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1995

Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.

Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.

Animals↗
PubMed1994

Detection of an unidentified potyvirus from Roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant DNA sequences.

Degenerate potyvirus-specific primers were used in the PCR to amplify cDNA representing a 335 nucleotide region of the coat protein gene in RNA purified from an uncharacterised potyvirus isolated from Roystonea regia palms in Queensland, Australia. The RNA was also detected by PCR in total nucleic acid extracts from infected Nicotiana benthamiana, N. clevelandii and Vicia faba. The method also successfully detected pea seed-borne mosaic virus in Pisum sativum. In addition the procedure amplified DNA's of approximately 200 bp and 420 bp, of non-viral origin, from total nucleic acid extracts of healthy Nicotiana spp, indicating that size of the PCR products needs to be included as a criterion for identifying virus specific products when this method is used.

Base Sequence↗
PubMed1993

A baculovirus encoded 16-kDa glycoprotein localizes near the nuclear membrane of infected cells.

An open reading frame (ORF 2) located upstream of the polyhedron envelope protein gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) was cloned in frame into a trpE expression vector. The fusion protein produced by this construct was used for the production of a monospecific antiserum. Western blot analysis of OpMNPV-infected Lymantria dispar cells detected a 16-kDa protein at 24 hr postinfection. The 16-kDa protein was determined to be N-glycosylated by tunicamycin treatment of infected cells. Immunofluorescence microscopy localized the 16-kDa protein to foci of intense cytoplasmic staining near the nuclear membrane. Immunoelectron microscopy indicated that the 16-kDa protein is associated with lamellar-like structures peripheral to the nuclear membrane and with envelopes of virus that have budded into the cytoplasm. The 16-kDa protein was not associated with extracellular budded or polyhedron-derived virions.

Animals↗
PubMed1993

Identification and characterization of a putative origin of DNA replication in the genome of a baculovirus pathogenic for Orgyia pseudotsugata.

A four-kilobase (kb) region (HindIII-N, map units 7.0-11.3) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. The plasmid DNA appeared to be replicated to form high molecular weight multimers. Plasmids with deletions of up to 1.8 kb from either end of the HindIII-N region were replication competent. However, a discrete sequence, contained within the region bracketed by the deletions, capable of specifying replication was not identified. No evidence for sequence homology was found between the OpMNPV HindIII-N region and regions elsewhere in the OpMNPV genome or to putative Autographa californica MNPV (AcMNPV) replication origins. Origin-dependent plasmid replication was shown to require the presence of the OpMNPV DNA polymerase gene. The OpMNPV origin replicated poorly in AcMNPV-infected Spodoptera frugiperda cells and, conversely, a putative AcMNPV origin (hr2) replicated at low levels in OpMNPV-infected Lymantria dispar cells.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence, serology and symptomatology suggest that vanilla necrosis potyvirus is a strain of watermelon mosaic virus II.

Vanilla necrosis potyvirus (VNV) is the cause of significant losses to the South Pacific islands vanilla crop. The gene for the coat protein of VNV has been cloned and sequenced. Comparison of this gene with other potyviral coat protein sequences revealed 97% nucleotide sequence homology (98% amino acid homology) to a US isolate of watermelon mosaic virus II (WMV-II), 93% nucleotide sequence homology (96% amino acid homology) to an Australian isolate of WMV-II and 81% nucleotide sequence homology (88% amino acid homology) to soybean mosaic virus-N (SMV-N). Serological analysis, by Western blot and ELISA, confirmed the close relationship between VNV and WMV-II. Furthermore, a limited host range determination found VNV and WMV-II able to infect the same series of test plants. However, symptoms differed significantly on three test species demonstrating that VNV and WMV-II are not identical in biological properties. We suggest that VNV be renamed WMV-II (Tonga).

Amino Acid Sequence↗
PubMed1993

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.

Amino Acid Sequence↗
PubMed1991

Immunoelectron microscopic examination of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus-infected Lymantria dispar cells: time course and localization of major polyhedron-associated proteins.

Immunoelectron microscopy was employed to examine the temporal expression and localization of two proteins involved in baculovirus polyhedron assembly (polyhedrin and p10) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) in infected Lymantria dispar cells. In addition, the association of p10 with the polyhedron envelope (PE) protein was studied. The major capsid protein (p39) was also examined to investigate the association of virion structural proteins with polyhedron formation. In infected cells, p39 did not show a concentrated association with any infected-cell structures other than nucleocapsids and appeared to be randomly distributed over the nucleocapsid surface. Likewise, polyhedrin showed no major concentrations outside of developing or mature polyhedra. The p10 antibody cross-reacted with a protein associated with condensed chromosomes in uninfected cells. In infected cells, p10 is a component of the body of fibrillar structures. The PE protein has been shown to accumulate around the periphery of fibrillar structures. Cells infected with a polyhedrin-minus virus expressing the beta-galactosidase gene under the control of the polyhedrin promoter were examined to determine whether the lack of polyhedra would influence the localization of major polyhedron-associated viral proteins. High concentrations of PE protein accumulating on the periphery of fibrillar structures appeared to be the major difference from wild-type virus-infected cells. The beta-galactosidase protein appeared to be distributed throughout the nucleus and cytoplasm, in contrast with the specific localization of the viral proteins.

Animals↗
PubMed1990

A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata: genetic location, sequence, transcriptional mapping, and immunocytochemical characterization.

Two lambda gt11 clones containing overlapping DNA inserts encoding portions of a structural protein gene from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) were identified by their immunoreactivity with polyclonal antisera produced against purified polyhedra-derived virus. Sequence analysis of a 3.6-kb region of the baculovirus genome (map units 69.1-71.6) from which the lambda gt11 inserts originated revealed an open reading frame of 1872 nt (624 amino acids) encoding a predicted protein of 70.6 kDa. Northern blot, primer extension, and 3' S1 analysis of this ORF indicated that an mRNA of approximately 2100 nt was transcribed from this gene. The mRNA appears to initiate from a late promoter/mRNA start site consensus sequence GTAAG and is expressed at late times postinfection. A gene fusion containing the C-terminal 368 amino acids of the gene was constructed using a bacterial trpE expression vector. Rabbit antiserum made against the purified fusion protein reacted with a protein of 87 kDa on Western blots of infected cell extracts at 24 hr p.i. and thereafter. The p87 protein was shown to be a component of both budded and polyhedra-derived virus and purified capsids. Immunofluorescence analysis indicated that p87 is expressed late in infection and concentrated in infected cell nuclei.

Amino Acid Sequence↗
PubMed1989

A baculovirus polyhedral envelope-associated protein: genetic location, nucleotide sequence, and immunocytochemical characterization.

Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), two immunoreactive lambda gtII clones were identified which contained nonoverlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hr postinfection (hr p.i.). The protein was observed in the cytoplasm and nucleus at 24 hr p.i. and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein of AcMNPV, pp34, recently reported by M.A. Whitt and J.S. Manning [(1988) Virology 163, 33-42].

Amino Acid Sequence↗