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C Grose

Publications and source records attributed to C Grose.

At least 19 recordsLinked to original sources
PubMed2006

Genomic cartography of varicella-zoster virus: a complete genome-based analysis of strain variability with implications for attenuation and phenotypic differences.

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.

Base Sequence↗
PubMed2006

Varicella zoster virus: out of Africa and into the research laboratory.

This review updates on numerous topics relating to the evolutionary origins of varicella zoster virus (VZV), the replication cycle, virion assembly and the recent genomic analyses. VZV is one of eight human herpesviruses that have existed for at least 400 million years. It has co-evolved with humankind and is present in all nationalities globally. The pathogenesis of varicella (chickenpox) is dependent on viral replication and dispersion through the body in T-lymphocytes. VZV replication is similar to that of herpes simplex virus. A complete analysis of VZV transcripts has identified their relative abundance, with transcripts for the regulatory proteins (open reading frame) ORF62 and ORF63 among the greatest. Studies of virion assembly have shown that endocytosis pathways are involved in the envelopment process by the viral glycoproteins. The complete sequencing of five VZV strains has identified numerous single nucleotide polymorphisms, and in turn, VZV strains have been segregated into European/North American and Asian clades. Furthermore, a small number of mutant VZV strains have been identified. These results suggest more diversity between VZV strains than previously recognized.

Animals↗
PubMed2003

Genomic analysis of varicella-zoster virus: primers for individual open reading frames.

The genome of varicella-zoster virus (VZV) contains nearly 125,000 bp. Preliminary genomic analysis has revealed that VZV may be less immutable than once thought. Through the investigation of the VZV genome using specifically designed oligonucleotides, it has been learned that sequence variation within VZV open reading frame 62 can distinguish between vaccine and wild-type virus. Additionally, the presence of single nucleotide polymorphisms within the VZV genome has identified distinct VZV populations originating from circumscribed geographic locations. In order for future studies of VZV genetic diversity to be carried out, amplifying and sequencing primers for individual VZV genes have been catalogued. Additionally, this report will facilitate the selection of VZV primers by which to distinguish clinical VZV isolates from vaccinia virus isolates.

DNA Primers↗
PubMed2003

Partner's influence on patient preference for treatment in early prostate cancer.

OBJECTIVE: To determine the partner's influence on the patient's choice of treatment for early prostate cancer, and whether partner characteristics and biases predict the preference. PATIENTS, SUBJECTS AND METHODS: Questionnaires for partners to complete retrospectively were sent to consecutive patients recruited in a study comparing treatment options for early prostate cancer. The partners' perceptions about prostate cancer were explored and the partners asked to comment on the suitability of each treatment option. Partners recorded their influence on the patient's choice using a 10-point visual linear analogue scale. RESULTS: Questionnaires were sent to 116 eligible patients and 82 were returned for analysis (mean partner age 63 years). When asked to recall the treatment options initially discussed, all partners recalled radiotherapy (EBRT), all but one radical prostatectomy (RP), 51% brachytherapy, but only 29% watchful waiting (WW); 41% of partners stated RP as their chosen option, 37% EBRT, 12% brachytherapy and 10% no clear favourite. None preferred WW. Employment and education status were not significant predictors of partners' preference but retired partners and those aged > 65 years were 3 times more likely to prefer EBRT than were their employed and younger counterparts, respectively. The partners' mean (median, SD) self-assessed influence factor was 4.8 (5, 3.4). Of the partners, 88% reported active involvement throughout the process, identifying information-gathering and emotional support as their primary roles. Most deliberately chose not to influence the patient's final decision. CONCLUSION: Partner preference is influenced by pre-existing conceptions about cancer and its treatment. While undoubtedly influential throughout the decision-making process, partners deliberately left the final decision to the patient.

Adult↗
PubMed2001

Varicella-zoster virus with a lost gE epitope: evidence for immunological pressure by the human antibody response.

The varicella-zoster virus (VZV) genome contains about 70 open reading frames (ORF). ORF 68 codes for glycoprotein gE, formerly called gpl, which is the predominant VZV glycoprotein; gE is a typical type 1 transmembrane protein with 623 amino acids. Recently, a variant virus was discovered which has a mutation in gE codon 150; this mutation converts an aspartic acid into an asparagine residue.

Adult↗
PubMed2001

Interactions among structural proteins of varicella zoster virus.

Varicella zoster virus tegument components include the regulatory proteins IE4, IE62, IE63 and the ORFI0 protein, a protein kinase (ORF47) and an abundant protein encoded in ORF9 which is the homolog of HSV VP22. The kinase is able to phosphorylate IE62 and the ORF9 protein specifically in viral particles. We show that interactions among these proteins are, at least in part, dependent on the presence or absence of phosphate groups and we suggest models for tegument formation and for its dissolution in the infected cell.

Animals↗
PubMed2001

Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not.

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

Animals↗
PubMed2001

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

Animals↗
PubMed2001

Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63.

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.

Aspartic Acid↗
PubMed2001

Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression.

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH+gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.

Antigens, Viral↗
PubMed2000

Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice.

Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses. In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress. Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture. This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal. For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker. By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain. Titrations by infectious center assays supported the above image analysis data. Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants. Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days. To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE). Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain. In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice.

Animals↗
PubMed1999

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase.

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

Amino Acid Sequence↗
PubMed1998

Antigenic variation of varicella zoster virus Fc receptor gE: loss of a major B cell epitope in the ectodomain.

Varicella zoster virus (VZV) is considered to possess a genetically stable genome; only one serotype is recognized around the world. The 125-kbp genome contains approximately 70 open reading frames. One that has received particular attention is open reading frame 68, which codes for glycoprotein gE, the predominant 623-residue viral envelope product that harbors both B and T cell epitopes. This report describes the initial characterization of a community-acquired VZV isolate that was a distinguishable second serotype (i.e., it had lost a major B cell epitope defined on the gE ectodomain by a murine monoclonal antibody called mAb 3B3). The mAb 3B3 epitope was found not only on the prototype sequenced Dumas strain from Holland and all previously tested North American isolates but also on the varicella vaccine Oka strain originally attenuated in Japan. Sequencing of the mutated gE ectodomain demonstrated that codon 150 exhibited a single base change that led to an amino acid change (aspartic acid to asparagine). Observation of the monolayers infected with the mutant VZV strain also led to the surprising discovery that the topography of egress was altered. Wild-type VZV emerges along distinctive viral highways, whereas the mutant strain virions were nearly uniformly distributed over the cell surface in a pattern more closely resembling egress of herpes simplex virus 1. The mutant VZV strain was designated VZV-MSP because it was isolated in Minnesota.

Amino Acid Sequence↗
PubMed1998

Varicella-zoster virus glycoprotein gE: endocytosis and trafficking of the Fc receptor.

Varicella-zoster virus (VZV) encodes a functional cell membrane Fc receptor called glycoprotein gE. VZV gE resembles other mammalian cell membrane receptors, such as the mammalian Fc receptor. In further analyses by transient transfection, the cellular trafficking of VZV gE was compared to other cell surface receptors. VZV gE was shown to undergo endocytosis from the cell membrane when visualized by laser scanning confocal microscopy. The endocytosis and trafficking pathway of VZV gE followed closely the pathway defined for the human transferrin receptor. Receptor-mediated endocytosis of VZV gE was dependent on a YAGL motif in its cytoplasmic tail. In addition, VZV gE underwent receptor-mediated endocytosis when it bound the Fc portion of immunoglobulin. Thus, this detailed study of VZV gE cellular trafficking has revealed potential roles for gE during viral infection.

Amino Acid Sequence↗
PubMed1998

Complex formation facilitates endocytosis of the varicella-zoster virus gE:gI Fc receptor.

Open reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor Fc gammaRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042-4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, approximately 32%) or gI alone (steady state, approximately 45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex.

Capsid↗