Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Expression of functional recombinant antibody molecules in insect cell expression systems.

Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein.

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterranea.

A monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3' half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immunoblotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.

Detection of potato mop-top virus capsid readthrough protein in virus particles.

Potato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was doned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV-infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.

Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus.

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.

Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.

Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene. PMTV CP was expressed in large amounts in some of the primary transformants. The five transgenic lines which produced the most CP were selected for resistance testing. Flowers on transformed plants were allowed to self-fertilize. Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV. Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated. T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated. Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea. In these tests only two plants out of 99 became infected. Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

Sequence analysis and gene content of potato mop-top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses.

The complete sequence of the 2315 nucleotides in RNA 3 of potato mop-top furovirus (PMTV) isolate T was obtained by analysis of cDNA clones and by direct RNA sequencing. The sequence contains an open reading frame for the coat protein (20K) terminated by an amber codon, followed by an in-phase coding region for an additional 47K. PMTV therefore resembles soil-borne wheat mosaic (SBWMV) and beet necrotic yellow vein (BNYVV) viruses (two other fungus-transmitted viruses with rod-shaped particles) in having a coat protein-readthrough product. Comparison of the 3' untranslated regions of PMTV RNA 2 and RNA 3 reveals a long conserved block of 150 nucleotides, which contains two repeated sequences and has the potential to form consecutive pseudoknot structures. PMTV RNA 3 ends a few nucleotides downstream of this conserved block, but RNA extends for a further 140 nucleotides, which can potentially form a 3'-terminal tRNA-like structure similar to those in the RNA species of SBWMV, tymoviruses, and some tobamoviruses. PMTV coat protein has amino acid sequence identities of 30 and 28% with SBWMV and BNYVV coat proteins, respectively, and apparent structural similarities with tobacco mosaic virus coat protein. The coat protein readthrough domains of PMTV, SBWMV, and BNYVV have shared residues throughout their length but no extended sequences are conserved. The presence in RNA 3 of coding sequences for only the coat protein and readthrough domain distinguishes PMTV from SBWMV and BNYVV, both of which have them in RNA 2 along with one or more other genes. Comparison of the genomes of PMTV, BNYVV, and SBWMV shows that furoviruses exhibit considerable heterogeneity in genome organization.

The nucleotide sequence of potato mop-top virus RNA 2: a novel type of genome organization for a furovirus.

Particles of isolate T of potato mop-top furovirus (PMTV) contain three RNA species (6.5, 3.0 and 2.5 kb). Hybridization tests with cloned cDNA probes showed that none of these species was derived from another. RNA 2 (2962 nt), which was sequenced, has non-coding regions of 368 nt and 285 nt at the 5' end and 3' end, respectively. Near the 5' terminus, nucleotides 46 to 110 are able to form a stem-loop structure, the stem of which has 23 bp with only one mismatch and one unpaired nucleotide. From the 5' end, the four open reading frames encode proteins of 51K, 13K, 21K and 8K. The first three of these have sequence similarity to the triple-gene-block proteins of other viruses, particularly barley stripe mosaic hordeivirus. The 51K protein contains a putative NTP-binding motif and the 13K and 21K proteins each contain two hydrophobic regions separated by a hydrophilic region. The 8K protein is rich in cysteine. PMTV differs from other furoviruses in having a tripartite genome. Its RNA 2 differs in gene content from the RNA 2 of soil-borne wheat mosaic virus, which lacks a triple gene block, and from that of beet necrotic yellow vein virus, which has a coat protein gene and read-through domain to the 5' side of its triple gene block. The gene arrangement in PMTV is therefore novel for a furovirus.

Enhancement of resistance to potato leafroll virus multiplication in potato by combining the effects of host genes and transgenes.

Four potato clones with host gene-mediated resistance to potato leafroll virus (PLRV) multiplication were transformed with the PLRV coat protein (CP) gene. Plants of lines expressing high levels of transcript were highly resistant to PLRV multiplication; virus concentration was only 20-40 ng/g of leaf, which is approximately 1% of the concentration reached in susceptible cultivars. The effects of the transgenic and host-derived resistance genes appear to be additive.

Sequence analysis of the parsnip yellow fleck virus polyprotein: evidence of affinities with picornaviruses.

The 9.9 kb monopartite ssRNA genome of parsnip yellow fleck virus (PYFV) encodes a polyprotein from which the functional proteins are assumed to arise by proteolytic cleavage. The 22.5K, 26K and 31K particle proteins were mapped in the polyprotein by determining their N-terminal amino acid sequences, and were found to begin at amino acid positions 395, 589 and 811, respectively. There could be polypeptide(s) of up to 43K on the N-terminal side of the particle protein sequences. A region within the 26K particle protein has sequence similarity to the VP3 particle protein of picornaviruses. Three other regions in the PYFV polyprotein have sequence similarity to regions thought to have RNA polymerase, NTP-binding and protease functions in the polyproteins of picornaviruses, comoviruses and nepoviruses. Despite these similarities in sequence and in genome organization to viruses in the picorna-like supergroup, PYFV is distinct from all other plant and animal viruses described. This justifies placing it in a separate plant virus genus for which the name 'sequivirus' has been proposed.

Parsnip yellow fleck and rice tungro spherical viruses resemble picornaviruses and represent two genera in a proposed new plant picornavirus family (Sequiviridae).

Parsnip yellow fleck and rice tungro spherical viruses, with monopartite ss RNA genomes, resemble picornaviruses in the polymerase and NTP-binding domains of their encoded polyproteins. Though in separate genera, they may comprise a new family.

Relationship between transcript production and virus resistance in transgenic tobacco expressing the potato leafroll virus coat protein gene.

The coat protein (CP) gene of potato leafroll luteovims (PLRV) was inserted into tobacco (Nicotiana tabacum) using disarmed Agrobacterium tumefaciens (LBA4404) containing a binary expression vector. PLRV CP gene transcript was detected in transgenic plants but its abundance differed between transformed lines. CP was not detected in virus-free transgenic plants. The segregation of kanamycin resistance in S1 seedling progenies (obtained by selfing transformed plants) indicated that multiple (up to five) integration events involving vector T-DNA had occurred in most transformants. However, the amount of detectable CP transcript was not related to the neomycin phosphotransferase II gene copy number. Multiplication of PLRV in mature transgenic plants was diminished by up to 6-fold; the greatest diminution was in those transformed lines in which most CP gene transcript was detected. However, S1 progeny seedlings of transgenic plants were no more resistant to infection, following inoculation with viruliferous aphids, than seedlings of non-transformed control plants.

The nucleotide sequence of parsnip yellow fleck virus: a plant picorna-like virus.

The complete sequence of 9871 nucleotides (nts) of parsnip yellow fleck virus (PYFV; isolate P-121) was determined from cDNA clones and by direct sequencing of viral RNA. The RNA contains a large open reading frame between nts 279 and 9362 which encodes a polyprotein of 3027 amino acids with a calculated M(r) of 336212 (336K). A PYFV polyclonal antiserum reacted with the proteins expressed from phage carrying cDNA clones from the 5' half of the PYFV genome. Comparison of the polyprotein sequence of PYFV with other viral polyprotein sequences reveals similarities to the putative NTP-binding and RNA polymerase domains of cowpea mosaic comovirus, tomato black ring nepovirus and several animal picornaviruses. The 3' untranslated region of PYFV RNA is 509 nts long and does not have a poly(A) tail. The 3'-terminal 121 nts may form a stem-loop structure which resembles that formed in the genomic RNA of mosquito-borne flaviviruses.

A tissue-type plasminogen activator mutant with prolonged clearance in vivo. Effect of removal of the growth factor domain.

The complete cDNA for human tissue-type plasminogen activator (t-PA) was cloned and sequenced. A mutant was constructed by using in vitro site-specific mutagenesis to delete the region encoding the growth factor domain (amino acids 51-87 inclusive). Normal and mutant t-PA species were produced using two mammalian expression systems (in human HeLa cells and mouse C127 cells). The clearance of mutant and normal t-PA from plasma was examined in vivo using a guinea pig model. Mutant t-PA derived from HeLa or C127 cells was cleared much more slowly than the cognate normal t-PA. The potential role of the growth factor domain in the recognition of t-PA by the hepatic clearance mechanism is discussed.

The gene organisation of a small RNA-containing insect virus: comparison with that of mammalian picornaviruses.

The coding regions of an insect virus, cricket paralysis virus, have been mapped using pactamycin. The results suggest that the genome of this virus functions as a polycistronic mRNA, the structural proteins being encoded by the 5' end of the RNA in an order similar to those of mammalian picornaviruses. High-molecular-weight proteins of unknown function map at the 3' end of the genome.

Translational inhibition of heat-shock induced gene expression in picornavirus-infected Drosophila melanogaster cells.

Picornavirus infection of Drosophila melanogaster cells inhibited the appearance of heat-shock induced proteins. Examination of intracellular mRNAs revealed that those coding for heat shock proteins were present in a translationally competent form in infected cells. Inhibition of induced gene expression in infected Drosophila cells therefore involves, but is not necessarily solely mediated by, effects at the level of translation.

Cell-free translation of Drosophila C virus RNA: identification of a virus protease activity involved in capsid protein synthesis and further studies on in vitro processing of cricket paralysis virus specified proteins.

Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsid precursors of either of the viruses.

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate).

The Ouarzazate strain of Drosophila virus (DCV0) was grown in Drosophila melanogaster tissue culture cells, and [35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of approximately 30,000 as well as several minor proteins of higher molecular weight and a protein of approximately 10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of approximately 200,000. The evidence presented in this paper supports the inclusion of DCV0 in the Picornaviridae group.