Biomedical subjects

Kathleen L Hefferon

Publications and source records attributed to Kathleen L Hefferon.

7 recordsLinked to original sources

PubMed2006

Multi-tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation.

Mastreviridae, of the family geminiviridae, possess a monopartite genome and are transmitted by leafhoppers. Bean yellow dwarf dirus (BeYDV) is a mastrevirus which originated from South Africa and infects dicoyledenous plants, a feature unusual for mastreviridae. Previously, the nonstructural proteins Rep and RepA were examined with respect to their independent roles in BeYDV replication. This was achieved by placing both gene products under independent constitutive promoter control and examining their effects on replication-competent constructs. In the current study, Rep and RepA are examined further for their roles in regulating BeYDV gene expression using a series of replication-incompetent constructs. While both Rep and RepA are found to behave as equally potent inhibitors of complementary-sense gene expression, they differ considerably with respect to their abilities to transactivate virion-sense gene expression. Furthermore, RepA is identified as playing more than one role in this transactivation process. A nuclear localization domain is identified in Rep which is absent in RepA, and Rep-RepA interactions are examined under in vivo conditions. The study concludes with an investigation into the expression strategies of the BeYDV capsid protein.

Fabaceae↗

PubMed2005

Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues.

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

Amino Acid Sequence↗

PubMed2004

Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system.

Edible vaccines have been generated from both transgenic plants as well as from plant viral vectors. Here, we have taken the best attributions of both systems and designed a minimalized version of the bean yellow dwarf geminivirus (BeYDV)-based replicon consisting of the cis-acting elements required for BeYDV replication as a means to express foreign genes in a plant cell line. Replication can be switched on at high levels upon expression of the BeYDV Rep protein, and gene expression enhanced enormously. Construction of an expression cassette encoding a synthetic vaccine gene and analysis of expression levels of a vaccine protein in a plant cell line system are described.

Antigens, Bacterial↗

PubMed2004

Baculovirus late expression factors.

Autographa californica nuclear polyhedrosis virus, or AcMNPV, is the type member of the baculoviruses, a family of double-stranded DNA viruses with large circular genomes. The successive and concomitant expression of an assortment of early, late and very late genes is instrumental for successful baculovirus infection, and requires a switch from early dependence on a host cell-derived polymerase II to a novel virus-encoded RNA polymerase that is required for transcription later on in infection. A series of repetitive and highly conserved sequences known as homologous regions, or hrs, function both as origins of DNA replication as well as transcriptional enhancers of late gene expression. An array of AcMNPV genes produced early on in infection, known as late expression factors, or LEFs, are essential for both replication and late gene expression. In this review, an overview of baculovirus LEFs and their roles in viral replication and late gene expression is presented. The role of LEFs in determining baculovirus host range is described. Finally, we compare baculovirus replication and transcription machinery with other viral systems.

Baculoviridae↗

PubMed2003

Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication.

Bean yellow dwarf virus (BeYDV) is a mastrevirus specific for dicotyledenous hosts. It contains four ORFs encoding a movement protein, a coat protein, and two Rep gene products, Rep and RepA, which are encoded by two overlapping ORFs. In this study, the roles of Rep and RepA in regulating replication of the BeYDV-based replicon were investigated by uncoupling them and placing Rep and RepA each under constitutive promoter control. Constitutive expression of both Rep and RepA supported replication and enhanced gene expression. When a reporter plasmid containing the Rep gene in the context of its native promoter was supplemented with additional Rep protein, replication was enhanced but the increase in gene expression was found to be more modest. Furthermore, expression of constitutively expressed RepA alone was found to reduce replication of this reporter construct as well as delay BeYDV replication in general. The effect of a RepA mutant with an altered retinoblastoma-related-protein binding motif on the efficiency of BeYDV replication was also examined. This mutant was found to severely diminish replication efficiency. Finally, the relationship of BeYDV coat protein to virus replication and reporter gene expression was investigated. Addition of coat protein increased accumulation of single-stranded DNA and had a detrimental effect on reporter gene expression.

Capsid Proteins↗

PubMed2003

ORF98 of Autographa californica nucleopolyhedrosisvirus is an auxiliary factor in late gene expression.

Autographa californica nucleopolyhedrosisvirus (AcMNPV) is the type member of the family Baculoviridae. Gene expression of AcMNPV during virus infection is temporally regulated. A series of late expression factors (LEFs) are required for late gene expression to take place. A number of additional factors have also been shown to more modestly influence late gene expression. Using the LEF transient assay, we scanned the AcMNPV genome for such factors by replacing plasmids using the LEF genes with larger clones and then looked for increases in late gene expression using a reporter plasmid under the control of a late promoter. Using this approach, ORF98 was identified as having a stimulatory effect on late gene expression. The ability of ORF98 to influence early, late, and very late gene expression was established. Furthermore, tagged versions of ORF98 were localized to the nuclei of transfected cells and were shown to interact with each other as homo-oligomers. Potential roles of ORF98 in baculovirus infection are discussed.

Animals↗

PubMed2002

Reconstructing the replication complex of AcMNPV.

Baculoviruses are well known for their large, circular, double-stranded DNA genomes. The type member, AcMNPV, is the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle. The viral genes involved in DNA replication have previously been identified and their products are required for the activation of late gene expression. In this study, we FLAG- and HA-tagged the replication late expression factors of AcMNPV, examined their expression and functional activities by CAT assay and Western blot analysis, and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy. We found that all replication LEFs with the exception of P143 and P35 resided in the nucleus of transfected cells. We further investigated the interactions among various replication LEFs using both yeast two-hybrid and coprecipitation strategies. A summary of the interactive properties of the replication LEFs is presented and a model for a putative AcMNPV replication complex is offered.

Animals↗