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Biomedical subjects

R S Coffin

Publications and source records attributed to R S Coffin.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cell type specific repression of the varicella zoster virus immediate early gene 62 promoter by the cellular Oct-2 transcription factor.

The cellular transcription factor Oct-2.1 has previously been shown to repress the transactivation of the varicella zoster virus (VZV) immediate early gene promoter by viral transactivators but not to inhibit its basal activity. In the case of the related virus herpes simplex virus (HSV), the effect of Oct-2 on the IE promoters has been shown to be cell type specific and to differ between the different alternatively spliced forms of Oct-2. Here we show that as well as Oct-2.1, the Oct-2.4 and 2.5 isoforms which are expressed in neuronal cells can inhibit transactivation of the VZV immediate early promoter regardless of the cell type used. In contrast, all the isoforms of Oct-2 can inhibit basal activity of the VZV promoter in neuronal cells but not in other cell types indicating that this effect is cell type specific. These effects are discussed in terms of the differential regulation of latent infections with HSV or VZV in dorsal root ganglia.

Animals↗
PubMed1998

A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain.

A number of viral vectors are currently being evaluated as potential gene therapy vectors for gene delivery to the brain. As well as evaluating their ability to express a transgene for extended periods of time it is also essential to examine any cytotoxic immune response to such vectors as this may not only limit transgene expression but also cause irreparable harm. This work describes the effect of inoculating a gamma34.5 mutant of herpes simplex type 1 (1716lacZ) into the brain of different strains of rats and mice. Animals were monitored for weight loss and signs of illness, and their brains were evaluated for inflammation, beta-galactosidase expression and recoverable infectious virus. We report that there is (i) a powerful immune response consisting of an early non-specific phase and a later presumably T-cell-mediated phase; (ii) significant weight loss in some animals strains accompanied by severe signs of clinical illness and (iii) transient reporter gene expression in all animal strains examined. To be useful for gene therapy we suggest this virus requires further modification, it should be tested in several animal strains and the dose of virus used may be critical in order to limit damage.

Animals↗
PubMed1998

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

Animals↗
PubMed1998

Potential and limitations of a gamma 34.5 mutant of herpes simplex 1 as a gene therapy vector in the CNS.

Direct injection of viral vectors into the central nervous system has become a valuable technique for exploring the function of neurological systems and is a potential therapy for neural disease. To this end a number of herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into the brain. In this study a non-neurovirulent HSV 17+ mutant, variant 1716, deleted in the gamma 34.5 gene and expressing the marker gene lacZ under the control of the latency-associated transcripts promoter was injected stereotactically into the central nervous system of two strains of rat (AO and PVG). We show (1) that transgene expression was low at the site of injection, in the striatum, at all times studied (12 h to 30 days after injection); (2) dramatically more transgene expression was observed at distant sites which contain neurons projecting directly to the site of injection, with maximal expression at these sites being at 1-2 days; (3) immunostaining with a polyclonal anti-HSV antibody and with an antibody which detects a 65 kDa HSV DNA binding protein (the product of the UL42 gene of the virus) demonstrated that viral gene products could be detected at the injection site as early as 12 h and up to 1 week after injection. Moreover these could also be detected at several secondary sites not all of which have direct connections with the injection site. These findings suggest that gamma 34.5 negative vectors have potential for gene transfer but may require further attenuation to limit viral antigen expression before they can be used successfully for gene therapy in the brain.

Animals↗
PubMed1998

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Animals↗
PubMed1998

Gene transfer using a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo.

The design of recombinant HSV-1 vectors for delivery of transgenes to the central nervous system is undergoing constant development. Problems associated with the construction and use of such vectors include the requirement for detection of recombinant versus nonrecombinant virus in vitro and also the identification of transduced cells in vivo. This could be overcome by the insertion of reporter genes such as lacZ or green fluorescent protein (GFP) under a separate promoter to the transgene to be expressed. In this case, however, reporter gene expression does not necessarily confirm transgene expression as a separate RNA must be produced. This study reports the use of an encephalomyocarditis virus internal ribosome entry site (IRES) to enable the translation of two reporter genes from a single mRNA transcript driven by the same promoter within a disabled HSV vector, and discusses the potential advantages of this approach.

Animals↗
PubMed1998

Delivery of a constitutively active form of the heat shock factor using a virus vector protects neuronal cells from thermal or ischaemic stress but not from apoptosis.

The heat shock proteins (HSPs) are induced by stressful stimuli and have a protective effect. Different HSPs protect with different efficiencies against different stresses indicating that optimal protection would be obtained with a non-stressful agent which induced a range of HSPs. We have prepared a herpesvirus vector expressing a constitutively active mutant form of heat shock factor 1 (HSF1) which, unlike the wild-type form of this transcription factor, does not require stress for its activation. Upon infection of neuronal cells, this virus induced a more restricted range of HSPs than in non-neuronal cells. Infection with the virus protected neuronal cells against subsequent thermal or ischaemic stress in accordance with its ability to induce HSP70 expression but did not protect them against apoptotic stimuli. The mechanisms of these effects and their significance for the use of HSF to manipulate HSP gene expression is discussed.

Animals↗
PubMed1998

The TAATGARAT motif in the herpes simplex virus immediate-early gene promoters can confer both positive and negative responses to cellular octamer-binding proteins when it is located within the viral genome.

The TAATGARAT motif in the herpes simplex virus (HSV) immediate-early (IE) gene promoters plays a key role in their activation by the Oct-1-Vmw65 complex, but its role in mediating inhibitory effects of cellular octamer-binding proteins is less clear. We have used indicator viruses containing reporter constructs with different IE promoters driving a reporter beta-galactosidase gene within the viral genome to investigate this. We showed that deletion of the upstream IE promoter region containing the TAATGARAT motifs abolishes the inhibitory effect of the cellular octamer-binding proteins Oct-2.4 and Oct-2.5 on the viral IE promoter. This inhibitory effect can be restored by addition of a single TAATGARAT motif to the minimal promoter within the viral genome. Hence, the TAATGARAT motif can indeed mediate both positive and negative effects of cellular transcription factors when it is located within the viral genome.

Animals↗
PubMed1997

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors.

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.

Animals↗
PubMed1996

Beet pseudo-yellows virus is an authentic closterovirus.

We have previously used RT-PCR to amplify a 251 base pair fragment diagnostic for beet pseudo-yellows virus (BPYV) infection from several BPYV infected samples from Europe and the USA. This has now been shown by sequencing to represent a fragment of a heat shock protein (HSP)70 gene. Sequence analysis of the fragment revealed that it is closely similar to the C-terminal sequence of the HSP70 homologue gene of lettuce infectious yellows virus, a definitive closterovirus, and also has obvious sequence similarities to the HSP70 genes of the other previously sequenced closteroviruses citrus tristeza virus and beet yellows virus. This observation confirms that BPYV is an authentic closterovirus with a wide geographical distribution.

Amino Acid Sequence↗
PubMed1996

Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs.

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CA1 neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constitutive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

Animals↗
PubMed1996

Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors.

Herpes simplex virus 1 (HSV1), while usually thought of as neurotrophic, can also efficiently infect a wide variety of non-neuronal cell types and so might be developed as a vector for gene delivery to non-neuronal as well as neuronal cells. Here we have tested three different disabled HSV vectors for their ability to deliver a lacZ gene to primary cardiac myocytes and vascular smooth muscle cells in vitro, and used the most efficient virus to transfect the rat heart in vivo. We also assessed the degree of cytopathic effect of the various viruses on the cardiac myocytes in vitro by testing the effects on the frequency of beating in synchronously beating myocyte cultures. While an HSV mutant in which the essential immediate-early gene IE2 had been deleted gave high efficiency gene transfer to the cardiac myocytes in vitro and the rat heart in vivo, viruses in which ICP34.5 or ICP34.5 and VMW65 were inactive (and which were also unable to replicate in these cells) gave a much lower efficiency of gene transfer, mirroring the degree of cytopathic effect observed in the beating myocyte cultures. Gene transfer to the vascular smooth muscle cells was considerably less efficient than to the myocytes in all cases. These results indicate that while HSV may be inappropriate for highly efficient gene transfer to the arterial wall, efficient gene transfer can be achieved in the myocardium, and thus that HSV vectors may be suitable for the alteration of cardiac cell physiology in vivo.

Animals↗
PubMed1996

Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors.

Herpes simplex virus 1 (HSV1) ICP34.5 deletion mutants are avirulent upon inoculation of both the central and peripheral nervous systems of mice, but they replicate to near wild-type titres in a number of non-neuronally derived cell lines in culture. Thus these mutants might be suitable for development as safe vectors for gene transfer to the nervous system. However, the mechanism of this avirulent phenotype in neuronal cells is at present poorly understood, although it has been suggested that nonpermissive cells infected with these mutants may undergo apoptosis, the function of ICP34.5 being to prevent this response and to allow continued virus replication. If this were the case ICP34.5 null mutants might be unsuitable for gene transfer as infected cells would quickly die, limiting the expression of a transgene. Here we have inserted a beta-galactosidase marker gene into a nonessential gene of an HSV1 strain 17+ mutant in which ICP34.5 has been deleted and also into a second mutant in which the virion transactivator protein VMW65 is also inactive. While all the mutants grew to high titre in tissue culture, mice inoculated by the foot-pad or intracranial route at high titre remained healthy until the end of the experiment. Moreover, beta-galactosidase was expressed either in the brain or in the dorsal root ganglia, depending on the site of inoculation. This suggests that in vivo the absence of ICP34.5 does not prevent the expression of a transgene in neuronal tissue and indicates that non-neurovirulent mutants lacking this gene may be suitable for further development as safe vectors for gene therapy in vivo.

Animals↗
PubMed1995

The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress.

A mild thermal stress protects primary cultures of dorsal root ganglion (DRG) neurons against a subsequent lethal heat stress as well as to a lesser extent against a subsequent lethal ischaemia. In contrast, a mild ischaemic stress protects DRG neurons only against a subsequent severe thermal stress and not against severe ischaemia. A greater induction of heat shock protein (hsp) synthesis was observed in these cells following mild temperature stress compared to mild ischaemia. This suggests that the protective effect observed is dependent on hsp synthesis resulting in the observed cross-protective effect and does not involve a particular pre-stress specifically protecting against a subsequent, more severe application of the same stress. Moreover, a particular level of hsp induction produces a better protective effect against lethal heat stress than against lethal ischaemia.

Animals↗
PubMed1995

Altered dinucleotide content within the latently transcribed regions of the DNA of alpha herpes viruses--implications for latent RNA expression and DNA structure.

The alphaherpesviruses establish latent infections in sensory neuronal cells, during which a number of latency-associated RNA transcripts (LATs) are produced. The reasons for the production of the LATs, however, and the mechanism by which the LAT region of the genome remains active during latency are unknown. Here we have analysed alphaherpesvirus sequences in an attempt to assess any differences between latently active regions of the genomes and the genomes as a whole which might be necessary for this differential expression pattern or for the LAT function, whatever that might be. We show that the LAT regions of all of the four alphaerpesviruses examined exhibit a previously unidentified and marked increase in CC + GG dinucleotides within the region and that three of the four viruses examined also show a marked decrease in CG + GC dinucleotides. The CC + GG increase was shown to occur throughout the LAT regions of these viruses, but the GC + CG decrease was shown to be confined only to LATs which are thought to be expressed as introns, and also to the intron sequences of the IE1 genes (at least for herpes simplex viruses 1 and 2), which overlap the LAT region. Bovine herpesvirus, the virus which did not show the GC + CG reduction in its LAT region but did show the GG + CC increase, is thought not to express LATs by a mechanism involving splicing, again indicating that the CG + GC reduction may be an intron-related phenomenon and also further suggesting that the two effects of CG + GC decrease and GG + CC increase may be independent of one another. Possible reasons for these unusual dinucleotide frequencies within the latently active regions of the alphaherpesviruses relating to DNA and RNA structure and to LAT function are discussed.

Base Sequence↗
PubMed1995

The tobacco necrosis virus p7a protein is a nucleic acid-binding protein.

The two centrally located open reading frames (ORFs) of necroviruses may, by analogy with the similarly located and related ORFs of carmoviruses, be expected to have a function in virus movement in plants. In the case of tobacco necrosis virus (TNV) strain D these proteins both have a molecular mass of approximately 7 kDa and are thus known as p7a and p7b. We overexpressed p7a in Escherichia coli, separated it from bacterial proteins and renatured it on gels, and showed that p7a was able to bind single-stranded RNA and single-stranded DNA, but was unable to bind double-stranded DNA. These protein-nucleic acid complexes were stable at moderately high salt concentrations. Protein p7b could not be expressed in a number of bacterial systems. We speculate that in TNV, unlike some other viruses which encode a single movement protein with separate functional domains for RNA binding and plasmodesmatal targeting, p7a and p7b may respectively provide these functions on separate proteins.

Base Sequence↗
PubMed1994

The infectivity of dimeric potato yellow mosaic geminivirus clones in different hosts.

Head-to-tail dimeric clones of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV) were constructed. These constructs were infectious when inoculated onto Nicotiana benthamiana plants either as DNA or by agroinoculation and were also infectious for tomato plants by agroinoculation. The dimers were not infectious for potato plants following inoculation by either method. Symptom induction required both DNA A and DNA B but agroinoculation with DNA A alone resulted in virus spread in 30% of the inoculated N. benthamiana plants. Leaf disc explants of N. benthamiana, tomato and potato could all be infected by agroinoculation indicating that the method of delivery of the DNA to intact potato plants was unsuitable for successful inoculation rather than an inherent inability of the virus to replicate/spread in potato per se. Neither whole plants nor leaf discs of sugar beet supported the replication of PYMV DNA.

Base Sequence↗