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Biomedical subjects

M E Penfold

Publications and source records attributed to M E Penfold.

8 recordsLinked to original sources
PubMed1999

Cytomegalovirus encodes a potent alpha chemokine.

Cytomegalovirus is a widespread opportunistic pathogen affecting immunocompromised individuals in whom neutrophils may mediate virus dissemination and contribute to progression of disease. Recent sequence analysis suggests that genes absent or altered in attenuated strains may influence pathogenesis. We have found two genes, UL146 and UL147, whose products have sequence similarity to alpha (CXC) chemokines. UL146 encodes a protein, designated vCXC-1, that is a 117-aa glycoprotein secreted into the culture medium as a late gene product, where its presence correlates with the ability to attract human neutrophils. Recombinant vCXC-1 is a fully functional chemokine, inducing calcium mobilization, chemotaxis, and degranulation of neutrophils. High-affinity vCXC-1 binding is shown to be mediated via CXCR2, but not CXCR1. vCXC-1 exhibits a potency approaching that of human IL-8. As the first example of a virus-encoded alpha chemokine, vCXC-1 may ensure the active recruitment of neutrophils during cytomegalovirus infection, thereby providing for efficient dissemination during acute infection and accounting for the prominence of this leukocyte subset in cytomegalovirus disease.

Amino Acid Sequence↗
PubMed1998

Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.

Binding Sites↗
PubMed1997

Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis.

To characterize the formation of replication compartments in human cytomegalovirus-infected cells, and to determine the fate of newly synthesized DNA, we localized viral replication proteins and DNA synthesis at early and late times during infection. As expected, ppUL57 (single-stranded DNA binding protein) and ppUL44 (DNA polymerase processivity factor) both localized to replication compartments beginning at 48 hpi. BrdU was incorporated into viral DNA in these compartments that was found to mature into progeny virus based on our ability to chase the label into the cytoplasm and out of the cell over the ensuing 72-h period. Although the pattern of BrdU incorporation at early times (20 or 24 hpi) was punctate, and distinct from the replication compartment that formed later during infection, viral DNA synthesized at this time also matured into progeny virus during a chase. Interestingly, sites of ppUL57 localization did not overlap completely with sites of BrdU incorporation at early times. Products from the UL112-113 gene localized to subnuclear regions by 6 hpi, earlier than ppUL57. Between 12 and 24 hpi, both ppUL57 and ppUL44 joined UL112-113 gene products at sites that subsequently developed into replication compartments. When infection was carried out in the presence of phosphonoformate or ganciclovir, replication compartment formation was blocked. A viral mutant deficient in uracil DNA glycosidase, previously shown to exhibit a delay in the initial phase of DNA replication, also exhibited delayed formation of replication compartments. These results raise the possibility that subnuclear sites defined by UL112-113 localization orchestrate the assembly of the CMV replication compartment and implicate punctate sites of BrdU incorporation as sites of early viral DNA replication that precedes the formation of the replication compartment.

Cell Compartmentation↗
PubMed1996

Herpes simplex virus protein targets for CD4 and CD8 lymphocyte cytotoxicity in cultured epidermal keratinocytes treated with interferon-gamma.

In early recurrent herpetic lesions, CD4 T lymphocytes are the predominant infiltrating cells, and keratinocytes expressing major histocompatibility complex (MHC) class II antigens, induced by interferon-gamma (IFN-gamma), are the major site of herpes simplex virus (HSV) replication. IFN-gamma pretreatment of human keratinocytes in vitro reduced MHC class I antigen down-regulation by HSV-1 infection and induced expression of HLA-DR that was unaltered by subsequent HSV-1 infection. Incubation of these infected keratinocytes with phosphonoacetic acid (PAA) almost completely inhibited expression of four major HSV glycoproteins, although expression of early proteins was not affected. Weak CD8 T lymphocyte cytotoxicity against IFN-gamma-stimulated, HLA-DR-expressing HSV-1-infected keratinocytes was consistently directed to the immediate early/early proteins (all 9 patients tested) but against late proteins to a lesser degree (4/9 patients). However, CD4 T lymphocyte cytotoxicity was much greater and directed predominantly against late HSV-1 glycoproteins (all 9 subjects tested) in these cells.

Antiviral Agents↗
PubMed1994

Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus.

Antibodies, Monoclonal↗
PubMedDate not supplied

A review of genetic differences between limited and extensively passaged human cytomegalovirus strains.

The complete genetic content of human cytomegalovirus (HCMV) has been difficult to determine, since most strains studied in the laboratory have been extensively passaged in human fibroblast cultures which can change the genetic content as well as the biological properties of the virus. Approximately 13 kb of novel DNA sequences located near the right edge of the unique long (UL) component of the genome has been discovered in Toledo, clinical isolates and certain stocks of Towne. This region of novel sequence, designated the UL/b' region, encodes several interesting proteins including vCXC-1, a potent IL-8 homologue, and UL144, a member of the TNF receptor family. This region is missing from the prototypic laboratory variants of Towne and AD169. In contrast to Toledo and other low passage isolates which have relatively small repeats bracketing the UL component, the Towne and AD169 laboratory variants contain large (>10 kb) b/b' repeats. The large size of these repeats in AD169 and Towne appear to have arisen as compensation for the loss of sequences from the UL/b' region that existed in less passaged variants of these strains. Consequently, many of the haploid genes at the left edge of the prototypic wild-type (wt) UL component are diploid in AD169 and Towne. We hypothesise that this plasticity of the genome at the right edge of the UL component results from extensive passage and adaptation to replication in fibroblasts in vitro. Further work will be required to understand the complete genetic content of wt HCMV.

Amino Acid Sequence↗
PubMedDate not supplied

The interaction of human fetal neurons and epidermal cells in vitro.

The interaction of autologous human fetal neurons with epidermal cells was studied by culturing fetal dorsal root ganglia (DRG) in the center of a dual chamber system with epidermal explants in the outer chamber. The two chambers were separated by two concentric stainless steel annular rings adherent to the substratum by silicon grease and agarose. Axons from the DRG penetrated the agarose barrier, growing into the exterior chamber by 10 d in vitro (DIV) and extended past sparse peripheral fibroblasts to interact specifically with epidermal cells by 12 to 16 DIV. Scanning electron microscopy (SEM) showed single or multiple neuronal fascicles terminating on epidermal cells with spatular, veillike or bulbous axon termini. Transmission electron microscopy (TEM) showed fine axonal termini between epidermal cells, separated by an intercellular gap. The specificity of axonal targeting for epidermal cells rather than fibroblasts was also demonstrated by infecting the DRG with Herpes simplex virus type-1 (HSV-1). Specific anterograde transport of HSV-1 along axons to keratin-expressing epidermal cells was demonstrated by immunofluorescence and immunoperoxidase staining using monoclonal antibodies to viral glycoprotein D. This model allows the study of the mechanism of the specific interactions between neurons and epidermal cells analogous to those in fetal development and after cutaneous nerve regeneration.

Axons↗