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PubMed2002

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA.

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

Ascomycota↗

PubMed1985

Effect of measles virus antibodies on a measles SSPE virus persistently infected C6 rat glioma cell line.

Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

Animals↗

PubMed1989

The temperature sensitivity of the Sabin type 3 vaccine strain of poliovirus: molecular and structural effects of a mutation in the capsid protein VP3.

The growth of the Sabin strain of type 3 poliovirus is reduced at high temperatures compared to that of its virulent precursor strain Leon. Recombinant viruses have been generated from infectious cDNA clones and demonstrate that the temperature-sensitive (ts) phenotype is mainly attributable to a difference in residue 91 of the virion protein VP3. Examination of non-ts mutants derived in vitro or in vivo reveals the existence of second site mutations some of which are clearly able to suppress the ts phenotype. The location of residue 91 of VP3, and of a number of candidate suppressor mutations, in the atomic structure of the virion suggests that the ts phenotype may result in destabilization of the particle and that the suppressors may function by stabilizing specific interfaces. It is not yet clear whether the ts phenotype is expressed at the level of the particle or in the form of defects in assembly or uncoating of the virion, or all three.

Amino Acid Sequence↗

PubMed1991

The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus.

A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined. Four open reading frames (ORFs) for proteins of Mr greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon. Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap. Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2. Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots. A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of Mr 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses. ORF V0 showed no significant homology with ORFs present in any other geminiviruses. A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.

Amino Acid Sequence↗

PubMed1993

The putative zinc finger of a caulimovirus is essential for infectivity but does not influence gene expression.

Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

Amino Acid Sequence↗

PubMed1993

The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes.

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

Animals↗

PubMed1994

Inhibition of feline immunodeficiency virus gene expression and replication by alphaherpesvirus ICP4 homologues.

We investigated the effects of pseudorabies virus (PRV), herpes simplex virus type 1 (HSV-1), and equine herpesvirus type 1 (EHV-1) ICP4 homologues on feline immunodeficiency virus (FIV) long terminal repeat (LTR)-directed gene expression. This was done by using the transient expression chloramphenicol acetyltransferase (CAT) assay in Crandell feline kidney (CRFK) and Felis catus whole fetus 4 cells transfected with a chimeric FIV LTR-CAT reporter construct in combination with effector plasmids expressing the PRV, HSV-1 or EHV-1 ICP4 homologue. The experiments demonstrated that the ICP4 homologues could significantly inhibit FIV LTR-directed gene expression. Moreover, the ICP4 homologues also exhibited a marked inhibitory effect on FIV replication in CRFK cells cotransfected with an infectious molecular clone of FIV.

Animals↗

PubMed1994

Cis- and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat.

Nuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV) were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (-50 to -150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (-124) and ATF (-58) motifs was observed, with cross-competition between these sites. A strong footprint signal was also detected over a tandemly repeated C/EBP motif (-94, -86) and an adjacent weaker footprint was found to be specific for an NF1 motif (-72/-63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation of the LTR was assayed using constructs linked to a CAT reporter gene. The full-length FIV LTR was not significantly trans-activated. However, the expression of a deleted LTR construct lacking the AP-4/AP-1 site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by co-transfection with an infectious molecular clone of FIV (FIV-PPR). These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression, and suggest that virus-coded trans-activators acting through U3 may play a role in some cellular environments.

Animals↗

PubMed1994

Deletion of a single N-linked glycosylation site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion.

The transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus type 1 possesses four consensus sites (Asn-X-Ser/Thr) for the incorporation of N-linked sugars situated on the extracellular domain of the molecule. The purpose of this investigation was to determine the significance of each of these sites in relation to the structure and function of the viral envelope glycoprotein. Each of the four sites was removed by in vitro mutagenesis of gp160 sequence in the non-infectious viral clone pEVd1443, so that amino acids 616, 621, 642 and 679 were each changed from asparagine to serine. The effects of mutagenesis were assessed by syncytium assay after wild-type or mutant envelope clones had been transfected into CD4+ HeLa cells. Removal of the glycosylation site at position 642 resulted in the synthesis of precursor gp160 that was neither cleaved, to give gp120 and gp41, nor transported to the plasma membrane of transfected cells. A consequence of these events was that envelope mutant 642 failed to induce syncytia between neighbouring cells in which it had been expressed. The results of this study indicate that N-linked glycosylation of Asn-642 in the glycoprotein produced by the pEVd1443 expression system is necessary for the correct intracellular processing of gp160 to yield surface-expressed, fusogenic gp41.

Base Sequence↗

PubMed1994

Comparison of biological properties of feline immunodeficiency virus isolates using recombinant chimeric viruses.

The biological properties of homogeneous populations of feline immunodeficiency viruses derived from infectious, molecular clones of the TM1, TM2 and Petaluma strains were compared. Differences in infectivity for Crandell feline kidney (CRFK) cells, and in syncytium formation and replication kinetics in a feline T lymphoblastoid cell line (MYA-1 cells) were observed. To investigate the basis of these differences between the TM2 and Petaluma strains, we first compared the basal promoter activity of the long terminal repeat which is a highly divergent region, but no significant difference in activities was found in CRFK cells. We then constructed two recombinant chimeric clones which carry gag, pol, vif, and ORF A from the heterologous virus. From analyses using the chimeric clones, it was revealed that efficient virus growth in CRFK cells and MYA-1 cells was regulated by the gag, pol, vif and ORF A regions, whereas viral determinants of infectivity for CRFK cells, and syncytium formation and cytopathogenicity in MYA-1 cells, were located in the env region.

Animals↗

PubMed1994

Persistent infection of MT-4 cells by human immunodeficiency virus type 1 becomes increasingly likely with in vitro serial passage of wild-type but not nef mutant virus.

Our previous studies have shown that human immunodeficiency virus type 1 (HIV-1), with mutations in accessory genes such as vif, vpr or vpu, can generate persistent infection of MT-4 cells, whereas infection by wild-type or nef mutant HIV-1 causes extensive cell death. The possibility of generating a naturally attenuated form of HIV-1 with reduced cytopathogenicity in MT-4 cells was examined by in vitro serial passage of the wild-type and a nef mutant form of HIV-1, each derived from the infectious molecular clone pNL432. The ability to cause persistent infection was observed after four passages of wild-type HIV-1 with the frequency of persistence becoming progressively higher with serial passage. In contrast, persistent infection was not observed even after 50 passages of the nef mutant virus. Sequence analysis of the accessory gene loci in genomes recovered from the persistent infections caused by passaged virus revealed mutations in vif and vpr, but not in vpu. The processing of the Env precursor to mature forms was not modified in any of the passages of either wild-type or nef mutant HIV-1. However, when compared with acute infections caused by similarly passaged virus of both wild-type and nef mutant HIV-1, persistent infections by passaged wild-type HIV-1 showed a significant decrease in the cell surface expression and function of Env. Cell surface CD4 was only partially down-regulated on cells acutely infected with the passaged viruses, whereas on cells persistently infected with passaged wild-type HIV-1 it was completely down-regulated. These results suggest that, during serial passage of HIV-1, mutations accumulate at least in the accessory genes vif and vpr in parallel with a lesser interaction between cell surface Env and CD4 molecules, and lead to the generation of less cytopathogenic viruses capable of persistent infection. Our results also suggest an important role for the nef gene product in the generation of HIV-1 strains that are less cytopathogenic.

Amino Acid Sequence↗

PubMed1995

Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: comparative sequence analysis of capillovirus genomes.

The complete nucleotide sequence of citrus tatter leaf capillovirus (CTLV lily strain) was determined. It is 6496 nucleotides long, excluding the 3'-terminal poly(A) tract, and contains two putative overlapping open reading frames (ORFs). ORF1 (positions 37-6354) encodes a potential polyprotein of molecular mass 242 kDa. ORF2 (positions 4788-5750) codes for a 36 kDa protein. The 242 kDa polypeptide contains several non-structural protein domains (i.e. methyltransferase, NTP-binding helicase, papain-like proteinase and polymerase) and, at its C terminus, the putative coat protein. The N-terminal region of the 36 kDa protein displays sequence similarity to the cell-to-cell movement proteins of the '30 K superfamily'. Such a genome structure is conserved between CTLV and apple stem grooving capillovirus. Capped transcripts from a plasmid containing the complete sequence of CTLV, with a T7 RNA promoter, successfully infected Chenopodium quinoa plants and caused symptoms characteristic of CTLV. Uncapped transcripts were noninfectious.

Amino Acid Sequence↗

PubMed1996

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.

Amino Acid Sequence↗

PubMed1999

Potyvirus helper component-proteinase and coat protein (CP) have coordinated functions in virus-host interactions and the same CP motif affects virus transmission and accumulation.

Amino acid differences between helper component-proteinase (HC-Pro) and coat protein (CP) that are putatively associated with biological differences between isolates PVA-B11 and PVA-U of potato A potyvirus (PVA) were studied using an infectious cDNA clone of PVA-B11. Replacement of the entire CP gene of PVA-B11 with the CP gene of PVA-U reduced virus accumulation in tobacco 5-fold, to the level of PVA-U. In contrast, four simultaneous amino acid substitutions made in PVA-B11 HC-Pro (according to PVA-U HC-Pro) increased virus accumulation 2- to 4-fold. A single substitution (S7G) at the CP N terminus reduced virus accumulation 10-fold, but restored aphid-transmissibility of PVA-B11. Simultaneous mutation of HC-Pro and replacement of CP in B11 delayed systemic movement in tobacco and limited cell-to-cell movement in potato. These results imply coordinated functions of HC-Pro and CP in accumulation and movement of PVA, because the phenotypic effects caused by simultaneous mutation of the two genes were different from the expected 'sum' of phenotypic changes observed following mutation of only one gene at a time.

Amino Acid Substitution↗

PubMed2000

The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription.

Equine arteritis virus (EAV) (ARTERIVIRIDAE:) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

Animals↗

PubMed2002

A DNA vaccine containing an infectious Marek's disease virus genome can confer protection against tumorigenic Marek's disease in chickens.

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek's disease virus (MDV) was tested for its potential to protect against Marek's disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.

Animals↗

PubMed2002

Mapping of tissue tropism determinants in coxsackievirus genomes.

Genomic regions responsible for the different tissue tropisms of coxsackievirus A9 (CAV9) and coxsackievirus B3 (CBV3) in newborn mice were investigated using recombinant viruses. Infectious cDNA clones of CAV9, a virus known to infect striated muscle, and CBV3, affecting the central nervous system, pancreas, liver, brown fat and striated muscle, were used to generate chimeric viruses. In situ hybridization analysis of different tissues from mice infected with the recombinant viruses, constructed by exchanging the 5' non-coding region (5'NCR), structural and non-structural genes, demonstrated that the pancreo- and liver tropism map predominantly to CBV3 sequences within the capsid genes, evidently due to receptor recognition. Although the major neurotropism determinant in the CBV3 genome was in the capsid region, viruses containing the CAV9 capsid were also able to initiate infection in the central nervous system provided they contained the CBV3 5'NCR. The presence of the 5'NCR of CAV9 clearly enhanced muscle tissue tropism.

5' Untranslated Regions↗

PubMed2003

An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea.

Cryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable experimental system for studying fungal pathogenesis. Moreover, the development of infectious cDNA clones of C. parasitica hypoviruses, capable of attenuating fungal virulence, has provided the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies of C. parasitica, the authors have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA isolated from wild-type (virus-free) and hypovirus-infected C. parasitica strains. Plasmid DNA was recovered from individual transformants and sequenced from the 5' end of the insert. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual ORFs. An assessment of functional diversity present in this collection was achieved by using the BLAST software utilities and the NCBI protein database. Candidate genes were identified with significant potential relevance to C. parasitica growth, development, pathogenesis and vegetative incompatibility. Additional investigations of a 12.9 kbp genomic region revealed microsynteny between C. parasitica and both Neurospora crassa and Magnaporthe grisea, two closely related fungi. These data represent the largest collection of sequence information currently available for C. parasitica and are now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.

Amino Acid Sequence↗