scholarly journals Microarray Analysis of Host Cell Gene Transcription in Response to Varicella-Zoster Virus Infection of Human T Cells and Fibroblasts In Vitro and SCIDhu Skin Xenografts In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1268-1280 ◽  
Author(s):  
Jeremy O. Jones ◽  
Ann M. Arvin
Keyword(s):  
T Cells ◽  
Gene Regulation ◽  
Varicella Zoster Virus ◽  
Host Cell ◽  
Gene Transcription ◽  
Cell Types ◽  
Cellular Gene ◽  
Varicella Zoster ◽  
Human T Cells

ABSTRACT During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection.

scholarly journals Varicella-Zoster Virus ORF47 Protein Kinase, Which Is Required for Replication in Human T Cells, and ORF66 Protein Kinase, Which Is Expressed during Latency, Are Dispensable for Establishment of Latency

2003 ◽  
Vol 77 (20) ◽  
pp. 11180-11185 ◽  
Author(s):  
Hitoshi Sato ◽  
Lesley Pesnicak ◽  
Jeffrey I. Cohen
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Latent Infection ◽  
Viral Proteins ◽  
Parental Virus ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Human T Cells ◽  
Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) results in a lifelong latent infection in human sensory and cranial nerve ganglia after primary infection. VZV open reading frame 47 (ORF47) and ORF66 encode protein kinases that phosphorylate several viral proteins, including VZV glycoprotein gE and ORF32, ORF62, and ORF63 proteins. Here we show that the ORF47 protein kinase also phosphorylates gI. While ORF47 is essential for virus replication in human T cells and skin, we found the gene to be dispensable for establishment of latent infection in dorsal root ganglia of rodents. ORF66 protein is expressed during latency. Rodents infected with VZV unable to express ORF66 developed latent infection at a rate similar to that for the parental virus. ORF63 transcripts, a hallmark of VZV latency, were also detected in similar numbers of animals infected with the ORF47 and ORF66 mutants and with the parental virus. VZV mutants unable to express four of the six genes that do not have herpes simplex virus (HSV) homologs (ORFs 1, 13, 32, 57) were also unimpaired for establishment of latency. While a truncated HSV VP16 mutant was previously reported to be unable to establish latency in a mouse model, we found that VZV with a deletion of ORF10, the homolog of HSV VP16, was dispensable for establishment of latency. Thus, seven genes, including one expressed during latency, are dispensable for establishing latent VZV infection.

Highly Reproducible Engraftment of Chronic Lymphocytic Leukemia (B-CLL) Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 52-52 ◽  
Author(s):  
Peter Ebeling ◽  
Jan Duerig ◽  
Florian Grabellus ◽  
Ulrich Duehrsen ◽  
Siegfried Seeber ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cell Clone ◽  
Cell Types ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
In Vivo Model ◽  
Acute Leukemias ◽  
Human T Cells

Abstract In contrast to normal hematopoiesis and acute leukemias, research in CLL still is hampered by the lack of a reliable in vivo model for primary B-CLL. We here report highly reproducible engraftment of B-CLL cells, when 1x10^8 MNC derived from the peripheral blood of CLL patients were transplanted i.v and i.p. into NOD/SCID mice. So far, 14 different CLL samples were investigated in 41 mice. At weeks 4, 8 or 12 mice were sacrificed and bone marrow (BM), spleen, and peritoneal fluid (PF) were analyzed by FACS for human CD19/CD5/CD23/CD45 (B-CLL) cells and CD45/CD3/CD5 (T) cells. Additionally, HE- and immunostaining was performed on spleen sections. Analysis at week 4 revealed engraftment in NOD/SCID mice for 13/14 samples (spleen: 13/14, BM: 4/14, PF: 12/14). B-CLL cells were observed predominantly in the spleen (8.9±2.4% or 9.1±4.4x10^5 cells) and PF (19.0±4.4% or 3.4±1.8x10^5 cells) with much lower engraftment in BM (0.6±0.3% or 0.1±0.1x10^5 cells). Detection of B-CLL cells in peripheral blood could be obtained in 3/14 experiments. Also substantial engraftment of human T-cells was observed in 13/14 experiments (spleen: 13/14, BM: 8/14, PF: 11/14). T-cells engraftment was highest in the spleen (23.8±9.8% or 28.7±13.1x10^5 cells) and somewhat lower in PF (16.4±8.2% or 3.0±1.6x10^5 cells) and BM (7.3±3.8% or 2.9±1.1x10^5 cells). Subpopulation analysis revealed a CD4+ phenotype in 65, 59 and 72 % of T-cells within spleen, PF and BM, respectively. Noteworthy, immunohistological analysis of HE stained spleen sections of engrafted animals revealed a pseudofollicular infiltration with human CD45LCA+ cells along splenic arterioles. Within these pseudofollicles human B-CLL but also CD3+ T-cells were detected. Contribution of B-CLL and T-cells to individual follicles was highly variable ranging from 5–95% for both cell types. When engraftment was analysed separately for the i.p and the i.v. route, engraftment of transplanted cells in PF seemed to be depended on the i.p. route whereas splenic engraftment was obtained following i.v. as well as i.p. injection. Sustained B-CLL engraftment was seen after 8 weeks (spleen: 3.1±1.4% or 7.3±3.1x10^5 total cells; PF: 57.6±23.3% or 1.0±0.5x10^5 cells; n=3 mice) and 12 weeks (spleen: 1.4±1.3% or 0.3±0.3x10^5 cells; PF: 10.2±7.3% or 0.5±0.5x10^5 cells; n=2 mice). Thus, we have shown efficient engraftment of human B-CLL cells in the spleen and PF of NOD/SCID mice. This in vivo model should significantly help to understand B-CLL biology and to test novel therapeutic approaches. The observed pseudofolicular pattern of splenic infiltration supports the theory of T-cells creating a “microenvironment” sustaining the growth of the leukemic B cell clone.

scholarly journals Glycoprotein I of Varicella-Zoster Virus Is Required for Viral Replication in Skin and T Cells

2002 ◽  
Vol 76 (16) ◽  
pp. 8468-8471 ◽  
Author(s):  
Jennifer Moffat ◽  
Hideki Ito ◽  
Marvin Sommer ◽  
Shannon Taylor ◽  
Ann M. Arvin
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Human Skin ◽  
Deletion Mutant ◽  
Varicella Zoster ◽  
Glycoprotein I

ABSTRACT Varicella-zoster virus (VZV) glycoprotein I (gI) is dispensable in cell culture; the SCIDhu model of VZV pathogenesis was used to determine whether gI is necessary in vivo. The parental and repaired viruses grew in human skin and thymus/liver implants, but the gI deletion mutant was not infectious. Thus, gI is essential for VZV infectivity in skin and T cells.

scholarly journals Dissecting the Molecular Mechanisms of the Tropism of Varicella-Zoster Virus for Human T Cells

2016 ◽  
Vol 90 (7) ◽  
pp. 3284-3287 ◽  
Author(s):  
Nandini Sen ◽  
Ann M. Arvin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Varicella Zoster Virus ◽  
Molecular Mechanisms ◽  
Cell Mass ◽  
Surface Proteins ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Human T Cells ◽  
Skin Homing

Studies of varicella-zoster virus (VZV) tropism for T cells support their role in viral transport to the skin during primary infection. Multiparametric single-cell mass cytometry demonstrates that, instead of preferentially infecting skin-homing T cells, VZV alters cell signaling and remodels surface proteins to enhance T cell skin trafficking. Viral proteins dispensable in skin, such as that encoded by open reading frame 66, are necessary in T cells. Interference with VZV T cell tropism may offer novel strategies for drug and vaccine design.

scholarly journals ORF66 Protein Kinase Function Is Required for T-Cell Tropism of Varicella-Zoster Virus In Vivo

2006 ◽  
Vol 80 (23) ◽  
pp. 11806-11816 ◽  
Author(s):  
Anne Schaap-Nutt ◽  
Marvin Sommer ◽  
Xibing Che ◽  
Leigh Zerboni ◽  
Ann M. Arvin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Kinase Domain ◽  
Cell Surface Expression ◽  
Cell Tropism ◽  
Varicella Zoster ◽  
Protein Functions

ABSTRACT Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.

scholarly journals Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo

2005 ◽  
Vol 79 (8) ◽  
pp. 4819-4827 ◽  
Author(s):  
Hideki Ito ◽  
Marvin H. Sommer ◽  
Leigh Zerboni ◽  
Armin Baiker ◽  
Bunji Sato ◽  
...  
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Gene Product ◽  
Human Skin ◽  
Growth Kinetics ◽  
Melanoma Cells ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster

ABSTRACT Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaΔ35(#1) and rOkaΔ35(#2), and replaced ORF35 at a nonnative site, generating two rOkaΔ35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaΔ35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaΔ35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaΔ35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaΔ35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaΔ35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaΔ35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.

Varicella-Zoster-Virus-Specific CD4+ Cytotoxic T Cells as Tumor-Specific Effector Cells in Cancer Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1292-1292
Author(s):  
Silke Landmeier ◽  
Sibylle Pscherer ◽  
Bodo Eing ◽  
Cliona M. Rooney ◽  
Heribert Juergens ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Varicella Zoster Virus ◽  
Cancer Immunotherapy ◽  
Peripheral Blood ◽  
Cytotoxic T Cell ◽  
Dependent Manner ◽  
Varicella Zoster ◽  
Ifn Γ

Abstract Adoptive transfer of gene-engineered T cells expressing tumor antigen-specific chimeric receptors (chRec) is a promising tool in cancer immunotherapy. A major limitation is the failure of chRec to induce proliferative T cell responses, resulting in a rapid loss of function. To provide a strategy for reexpansion of tumor-reactive T cells in vivo, we generated dual-specific T cells that respond to varicella zoster virus while also possessing chRec-mediated tumor reactivity. We expanded VZV-specific cytotoxic T cell lines (VZV-CTL) from four seropositive donors by culturing peripheral blood-derived T cells with lysates extracted from VZV-infected fibroblasts. Repeated stimulation with VZV lysates resulted in efficient and continued expansion for 10–12 weeks. >1x109 T cells were routinely obtained from a starting number of 1x106 peripheral blood T cells. The T cells displayed a mainly CD3+CD4+ (90±5%) phenotype. ELISPOT assays showed specific, MHC class II-restricted IFN-γ release in response to CD40-activated B cells expressing the viral glycoproteins gE and IE62. VZV-CTL belong to a non-regulatory effector T cell subset, shown by their failure to exert antiproliferative effects against cocultured autologous T cells and lack of Foxp3 expression. Retroviral transduction with chRec recognizing the tumor ganglioside antigen GD2 (14.G2a-ζ) and the B cell lineage antigen CD19 (CD19-ζ) resulted in receptor surface expression on 29–74% and 39–45% of cells, respectively. Gene-modified VZV-CTL efficiently recognized antigen-expressing tumor targets in an MHC-independent manner, as demonstrated by antigen-specific secretion of IFN-γ in response to coincubation with GD2-expressing tumor targets. Furthermore, chRec-transduced VZV-CTL performed potent and antigen-specific tumor cytolysis. Antibody blocking experiments revealed that tumor cells were lysed in a granulysin-dependent manner. ChRec-transduced CD3+CD4+ cytolytic VZV-CTL may provide a source of highly potent tumor-reactive cells for adoptive immunotherapy cancer. Endogenous viral reactivations or administration of booster doses of varicella vaccine may lead to survival of these tumor-reactive T cells for prolonged periods of time in vivo.

scholarly journals Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells.

1986 ◽  
Vol 58 (1) ◽  
pp. 179-184 ◽  
Author(s):  
A R Hayward ◽  
O Pontesilli ◽  
M Herberger ◽  
M Laszlo ◽  
M Levin
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Specific Lysis ◽  
Varicella Zoster ◽  
Human T Cells

scholarly journals T-Cell Tropism and the Role of ORF66 Protein in Pathogenesis of Varicella-Zoster Virus Infection

2005 ◽  
Vol 79 (20) ◽  
pp. 12921-12933 ◽  
Author(s):  
Anne Schaap ◽  
Jean-Francois Fortin ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
Shaye Stamatis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Varicella Zoster Virus ◽  
Stop Codon ◽  
Varicella Zoster Virus Infection ◽  
Cell Tropism ◽  
Varicella Zoster ◽  
Efficient Replication

ABSTRACT The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-γ. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.

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