Biomedical subjects

S Efstathiou

Publications and source records attributed to S Efstathiou.

At least 37 records · Page 2Linked to original sources

PubMed1999

Gene transfer with herpes simplex vectors.

In developing any viral gene delivery vector there are two fundamental problems which need to be addressed. Firstly, replication disabled vectors which will be safe for clinical use must be constructed, and secondly, strategies for obtaining appropriate transgene expression in vector transduced cells must be devised. In this review, the progress which has been made in developing herpes simplex virus (HSV)-based gene delivery vectors is discussed, as are the experimental results which have been obtained using these vectors for gene delivery in tissue culture cells and animal models.

Animals↗

PubMed1998

Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of wild rodents and is genetically related to human herpesvirus 8 and Epstein-Barr virus. The ability of MHV-68 to establish acute and persistent infection within laboratory mice offers a unique opportunity to investigate immunological and virological aspects of gammaherpesvirus pathogenesis.

Animals↗

PubMed1998

High adenoviral loads stimulate NF kappaB-dependent gene expression in human vascular smooth muscle cells.

Replication-deficient adenoviral vectors have been widely used for gene transfer with the aim of delivering genes of interest to investigate their function and potentially to treat human disease. The ability to critically evaluate the biological role of a gene of interest, using adenovirus-based vectors, has been hampered by the development of local inflammation at the site of delivery. We have demonstrated that high multiplicity infection of human VSMCs with a replication-deficient adenoviral vector expressing no transgene leads to activation of the transcription factor NF kappa B. Activation of NF kappa B by this mechanism was able to augment gene expression from the human cytomegalo-virus immediate-early promoter (CMV-IEP) and induce expression of the adhesion molecule ICAM-1 in human VSMCs. These effects were inhibited by pretreatment with N alpha-p-tosyl1-L-lysine chloromethyl ketone (TLCK), a serine protease inhibitor known to inhibit the activation of NF kappa B. This important effect of the vector itself may have profound implications when replication-deficient adenoviral vectors are used for experimental gene transfer at a high multiplicity of infection.

Adenoviridae↗

PubMed1998

Disruption of the 5' and 3' splice sites flanking the major latency-associated transcripts of herpes simplex virus type 1: evidence for alternate splicing in lytic and latent infections.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are the only viral gene products expressed within latently infected neurones. The most abundant (major) LATs consist of two collinear nuclear polyA- RNAs of 2 kb and 1.5 kb which it has been suggested represent stable introns derived from a less abundant primary transcript (minor LAT). Consistent with this proposition is the identification of consensus splice donor and acceptor sites flanking major LATs which are conserved between HSV types 1 and 2. Here we test the functionality of the predicted splice sites within the context of the virus genome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were constructed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation during productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice donor site significantly reduces levels of 2 kb major LAT during productive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in situ hybridization analyses of latently infected neuronal tissue. From these data it can be concluded that the proposed splice sites flanking the major LAT region are dispensable for 2 kb major LAT production in neurones latently infected with HSV-1 but constitute functional splicing signals in productively infected non-neuronal cells.

Alternative Splicing↗

PubMed1998

An efficient selection system for packaging herpes simplex virus amplicons.

Due to their simplicity and flexibility of genomic construction, herpes simplex virus (HSV) amplicon-based vectors are attractive vehicles for gene delivery. However, a significant problem faced in the generation of amplicon stocks is the low amplicon to helper virus (A/H) ratio. In order to improve the proportion of amplicons generated, a selection system for amplicon production was developed in which the HSV thymidine kinase (TK) gene is inserted into an amplicon plasmid and an HSV mutant with both TK and glycoprotein H (gH) genes deleted is used as a helper virus. Using a protocol in which amplicon stocks are passaged 2-3 times in BHK cells of TK- and gH+ genotype in the presence of selection medium containing methotrexate, stock preparations with high A/H ratio (up to 5:1) and high amplicon titre (>1 x 10(9) infectious units/ml) were generated. In vitro characterization demonstrated that a high level of biologically functional products can be efficiently produced from these amplicon constructs.

Animals↗

PubMed1998

Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo.

Experimental infection of inbred strains of laboratory mice with murine herpesvirus 68 (MHV-68), a natural pathogen of wild rodents, results in acute productive infection of the lung followed by a latent infection of B lymphocytes. We have previously shown that MHV-68 encodes an open reading frame with similarity to poxvirus serpins, designated ORF1, and eight novel tRNA-like sequences. The latter are processed into mature, uncharged tRNAs and are abundantly expressed during both lytic and latent infection. In this study it is demonstrated that deletion of four of the tRNA-like sequences and ORF1 from the virus genome does not affect the ability of MHV-68 to replicate in vitro or to establish, and reactivate from, latency in vivo.

Animals↗

PubMed1997

The use of herpes simplex virus-based vectors for gene delivery to the nervous system.

The ability of herpes simplex virus (HSV) to establish a lifelong, latent infection within neurons has led to much interest in the development of HSV-based vectors for neuronal gene delivery. This review discusses the progress made towards the construction of safe, replication-disabled HSV vectors that are capable of directing long-term transgene expression in latently infected neurons. Such vectors are now being investigated in a variety of animal model systems, with a view to developing gene therapy approaches to a number of metabolic and degenerative neurological diseases.

Animals↗

PubMed1997

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Animals↗

PubMed1997

Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency.

Murine gammaherpesvirus 68 (MHV-68) is a virus of wild rodents and is a convenient small animal model for studies of gammaherpesvirus pathogenesis. We have sequenced 6162 bp at the left end of the MHV-68 genome and identified two unique open reading frames (ORFs) (ORF2 and ORF3) and an ORF (ORF1) which displays similarity to poxvirus members of the serpin family. Interspersed with the ORFs is a family of eight novel tRNA-like sequences sharing tRNA-like predicted secondary structures and RNA polymerase III promoter elements. These sequences are expressed to high levels during lytic infection and are processed into mature tRNAs with post-transcriptionally added 3' CCA termini, indicating their recognition as tRNAs by cellular machinery. Acidic Northern analysis of four tRNAs tested has demonstrated that they are not aminoacylated by aminoacyl-tRNA synthetases present in the infected cell. Thus, it is currently unclear what biological function these uncharged viral tRNA-like sequences may fulfil. In situ hybridization analysis has shown that in addition to being expressed within productively infected tissues during acute stages of infection, the tRNA-like sequences are abundantly expressed within splenic germinal centres of latently infected mice. Therefore, the MHV-68 viral tRNAs represent a marker for latent infection and constitute the first report of tRNA-like sequences encoded by a virus of eukaryotes.

Acylation↗

PubMed1997

Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system.

The ability of herpes simplex virus type 1 (HSV-1) to establish a lifelong, transcriptionally active, latent infection in neurons has led to much interest in developing HSV-based vectors for gene delivery to the nervous system. A prerequisite of such vectors is that they should be capable of directing long-term transgene expression in latently infected neurons. The continued transcription of HSV-1 latency-associated transcripts (LATs) during neuronal latency suggests that regulatory sequences which mediate expression of LATs could be utilized for long-term expression of heterologous genes in the mammalian nervous system. In addition to upstream regulatory elements which are important for LAT promoter-mediated transcription during neuronal latency, there is growing evidence that sequences downstream of the LAT transcription start site play an important role in facilitating long-term latent-phase transcription. In order to maintain the integrity of both upstream and downstream regulatory elements of the LAT promoter, we constructed viruses which contained the lacZ and lacZ-neo reporter genes linked to the encephalomyocarditis virus internal ribosomal entry site (IRES) (viruses LbetaA and LbetaB, respectively) inserted approximately 1.5 kb downstream of the LAT transcription start site. These viruses expressed low levels of beta-galactosidase in lytically infected Vero cells and in cervical dorsal root ganglion neurons during the acute stage of infection in vivo. In contrast, at later times postinfection and consistent with the establishment of latency, increases both in the numbers of neurons expressing beta-galactosidase and in the intensity of staining were observed. Examination of the brain stems and spinal cords of animals latently infected with LbetaA, sampled at time points from 72 to 307 days postinfection, revealed the stable expression of beta-galactosidase within neurons located in facial and hypoglossal nerve nuclei and the upper cervical spinal cord. We conclude that the insertion of an IRES linked to a reporter gene 1.5 kb downstream from the LAT transcription start site does not disrupt elements of the LAT promoter necessary for long-term gene expression and, in both the peripheral and central nervous systems, facilitates beta-galactosidase expression in a wide variety of neurons.

Animals↗

PubMed1996

In vivo complementation studies of a glycoprotein H-deleted herpes simplex virus-based vector.

The utilization of herpes simplex virus (HSV) as a vector for gene delivery to the nervous system or as a live vaccine delivery system is dependent on the construction and characterization of disabled virus mutants which are unable to cause disease. Under certain circumstances, however, replication-defective vectors may carry a potential risk if they can be efficiently complemented by a co-infecting wild-type virus. Stocks of defective vectors should, therefore, be free from replication-competent virus, and helper cell lines should be incapable of generating replication-competent virus by recombination between the vector and the complementary gene. We describe a glycoprotein H-negative (gH-) virus/helper cell line combination which generates helper-free defective virus stocks containing replication-competent virus at a frequency no higher than 1 in 10(9) p.f.u. This virus/helper cell system provides a suitable background for the construction of safe replication-defective gene delivery vectors. In vivo studies demonstrate that gH- virus is unable to initiate disease in mice and establishes latency at low efficiency compared to wild-type HSV. To determine whether gH- virus can be complemented by wild-type virus in vivo, mice were infected with a variety of mixtures of these viruses. Complementation was observed in a minority of animals infected with more than 10(6) p.f.u. of both wild-type and defective virus but the most common observation was that the presence of defective virus suppressed entry of wild-type virus into the nervous system.

Animals↗

PubMed1996

A murine RNA polymerase I promoter inserted into the herpes simplex virus type 1 genome is functional during lytic, but not latent, infection.

The development of herpes simplex virus as a vector for neuronal gene delivery is dependent upon the identification and characterization of promoter elements capable of driving long-term expression during latency. The majority of RNA polymerase II (pol II) promoters studied are active during acute infection but silenced during latency. In order to investigate the potential of a murine RNA polymerase I (pol I) promoter to drive reporter gene expression during lytic and latent infection, we describe the construction and characterization of two recombinant viruses; SC16 LAT neo and SC16 US5 neo. These viruses contain a pol I-encephalomyocarditis virus internal ribosome entry site (EMCV IRES)-neomycin phosphotransferase gene (neoR) cassette inserted into the non-essential major latency associated transcript (LAT) and US5 regions respectively. Pol I promoter activity could be detected in the rodent BHK cell line, but not the primate derived Vero cell line-- consistent with the known species specificity of such promoters. This activity was specific to a virus containing an active pol I promoter. However, in situ hybridization analyses of latently infected cervical dorsal root ganglia failed to detect pol I mediated transcription of the reporter gene indicating that the murine pol I promoter is silenced following the establishment of latency. Insertion of the pol I-EMCV IRES-neoR cassette into the major LAT locus resulted in the production of a hybrid LAT transcript during latency which was translocated to the cytoplasm of latently infected neurones.

Animals↗

PubMed1996

Enhancer stimulation unmasks latent gene transfer after adenovirus-mediated gene delivery into human vascular smooth muscle cells.

Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing beta-galactosidase (RAd35 beta-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-kappa B response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate beta-galactosidase expression in infected cells. After infection with RAd35 beta-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of beta-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.

Adenoviridae↗

PubMed1995

The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution.

The complete DNA sequence was determined for strain U1102 of human herpesvirus-6, a CD4+ T-lymphotropic virus with disease associations in immunodeficient settings and a possible complicating factor in AIDS. The genome is 159,321 bp in size, has a base composition of 43% G + C, and contains 119 open reading frames. The overall structure is 143 kb bounded by 8 kb of direct repeats, DRL (left) and DRR (right), containing 0.35 kb of terminal and junctional arrays of human telomere-like simple repeats. Since eight open reading frames are duplicated in the repeats, six span repetitive elements and three are spliced, the genome is considered to contain 102 separate genes likely to encode protein. The genes are arranged colinearly with those in the genome of the previously sequenced betaherpesvirus, human cytomegalovirus, and has a distinct arrangement of conserved genes relative to the sequenced gammaherpesviruses, herpesvirus saimiri and Epstein-Barr virus, and the alphaherpesviruses, equine herpesvirus-1, varicella-zoster virus, and herpes simplex virus. Comparisons of predicted amino acid sequences allowed the functions of many human herpesvirus-6 encoded proteins to be assigned and showed the closest relationship in overall number and similarity to human cytomegalovirus products, with approximately 67% homologous proteins as compared to the 21% identified in all herpesviruses. The features of the conserved genes and their relative order suggested a general scheme for divergence among these herpesvirus lineages. In addition to the "core" conserved genes, the genome contains four distinct gene families which may be involved in immune evasion and persistence in immune cells: two have similarity to the "chemokine" chemotactic/proinflammatory family of cytokines, one to their peptide G-protein-coupled receptors, and a fourth to the immunoglobulin superfamily.

AIDS-Related Opportunistic Infections↗

PubMed1995

Herpes virus-based vectors.

Herpesviruses are a diverse family of large DNA viruses, all of which have the capacity to establish lifelong latent infections. Many different herpesviruses may have potential as gene delivery vehicles, but exploitation of this potential has, to date, been explored only using Herpes simplex virus (HSV), a virus which naturally establishes a silent, latent infection of neurones in man and in a number of experimental animal models. Delivery of reporter genes in vitro and in vivo has been demonstrated using a variety of replication competent and replication defective vectors, and significant physiological modification in the CNS has been achieved by HSV-mediated gene delivery. Much remains to be done using animal models and, in particular, the requirements for long-term gene expression from latent virus genomes needs to be defined in different cell types in vivo.

Gene Transfer Techniques↗

PubMed1995

The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons.

Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares a similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.

Amino Acid Sequence↗

PubMed1994

Identification of novel herpes simplex virus replicative intermediates by field inversion gel electrophoresis: implications for viral DNA amplification strategies.

Many facets of herpes simplex virus (HSV) DNA replication are not understood and advances in our knowledge depend on accurate characterization of high-molecular-weight replicative intermediates. In the present work, we have used a refinement of field-inversion gel electrophoresis (FIGE) to analyze infected-cell DNA. Infected Vero cells were encapsulated and manipulated in agarose microbeads, allowing intact replicative intermediates to be recovered easily from the wells of FIGE gels after electrophoretic removal of 152-kb linear viral genomes. Digestion of replicative intermediates with SpeI, which cuts the viral genome once, generated two novel DNA fragments (186 and 118 kb), in addition to the expected unit-length fragment (152 kb) predicted to arise from head to tail concatemers generated by rolling-circle replication. The SpeI fragments are the products of previously unidentified concatemers containing a head to tail arrangement of different HSV isomers, with respect to the orientation of the long segment of the viral genome. Such concatemers were prominent at an early stage of DNA synthesis when replicating DNA appeared still to be in a circular configuration, raising the possibility that isomerization of the viral genome is intimately linked to the initial round of DNA replication. Moreover, high-molecular-weight replicative intermediates were flanked exclusively by the long segment of the viral genome, indicating a unique initiation/termination or cleavage/packaging mechanism during HSV DNA replication and viral maturation.

DNA, Viral↗

PubMed1994

Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains.

The relationship between herpes simplex virus (HSV) DNA replication and establishment of latent infection was examined using an experimental model that makes use of the segmental sensory innervation of mouse flanks (T7 to T12). Ganglia from consecutive thoracic segments of C57BL/10 mice latently infected with a virulent strain of HSV-1 (SC16) were compared with respect to (i) HSV DNA levels, (ii) latency-associated transcripts (LATs) and (iii) numbers of LAT+ neurons. In concordance with previous results, two patterns of virus persistence were detected distinguished by either a low (10 to 23) or high (approx. 200) number of viral genomes/LAT+ neuron. The high copy pattern was associated, anatomically, with ganglia directly innervating inoculated skin (T7/8). Paradoxically, the highest number of LAT+ neurons and the highest concentrations of LATs were detected in spinal segments (e.g. T10) containing the lowest number of viral genomes, implying that most of the latent SC16 DNA detected at T7 and T8 was transcriptionally repressed. When neuronal amplification of HSV DNA during the establishment phase was prevented by infecting mice with a viral thymidine kinase deletion mutant (TKDM21), the high copy pattern was eliminated and each LAT+ neuron contained, on average, 22 TKDM21 genomes. We conclude that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with SC16. Structurally, latent TKDM21 DNA lacked free genomic termini, consistent with persistence of input DNA in an integrated or circular episomal configuration.

Animals↗