Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription

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.

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Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication

Journal of Virology ◽

10.1128/jvi.01322-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11723-11733 ◽

Cited By ~ 11

Author(s):

Mohamed I. Khalil ◽

John Hay ◽

William T. Ruyechan

Keyword(s):

Dna Replication ◽

Varicella Zoster Virus ◽

Binding Sites ◽

Site Mutation ◽

Mobility Shift ◽

Varicella Zoster ◽

Cellular Transcription Factor ◽

Downstream Region ◽

Gc Box ◽

Cellular Transcription

ABSTRACT The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.

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A Sequence within the Varicella-Zoster Virus (VZV) OriS Is a Negative Regulator of DNA Replication and Is Bound by a Protein Complex Containing the VZV ORF29 Protein

Journal of Virology ◽

10.1128/jvi.05501-11 ◽

2011 ◽

Vol 85 (23) ◽

pp. 12188-12200 ◽

Cited By ~ 6

Author(s):

M. I. Khalil ◽

A. Arvin ◽

J. Jones ◽

W. T. Ruyechan

Keyword(s):

Dna Replication ◽

Varicella Zoster Virus ◽

Protein Complex ◽

Negative Regulator ◽

Varicella Zoster

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Viral and cellular gene transcription in fibroblasts infected with small plaque mutants of varicella-zoster virus

Antiviral Research ◽

10.1016/j.antiviral.2005.06.011 ◽

2005 ◽

Vol 68 (2) ◽

pp. 56-65 ◽

Cited By ~ 12

Author(s):

Jeremy O. Jones ◽

Ann M. Arvin

Keyword(s):

Varicella Zoster Virus ◽

Gene Transcription ◽

Cellular Gene ◽

Small Plaque ◽

Varicella Zoster

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Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2Pon Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

Journal of Virology ◽

10.1128/jvi.02617-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1231-1243 ◽

Cited By ~ 8

Author(s):

Heather H. Henderson ◽

Kensey B. Timberlake ◽

Zoe A. Austin ◽

Hussain Badani ◽

Bridget Sanford ◽

...

Keyword(s):

Rna Polymerase ◽

Varicella Zoster Virus ◽

Rna Polymerase Ii ◽

Quantitative Pcr ◽

Gene Transcription ◽

Chromatin Immunoprecipitation ◽

Transcript Abundance ◽

Varicella Zoster ◽

Virus Gene ◽

Dna Shearing

ABSTRACTRegulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5P) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2P)-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi.IMPORTANCERegulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5Poccupancy decreased and S2Plevels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.

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Transcriptomal analysis of varicella-zoster virus infection using long oligonucleotide-based microarrays

Journal of General Virology ◽

10.1099/vir.0.80946-0 ◽

2005 ◽

Vol 86 (10) ◽

pp. 2673-2684 ◽

Cited By ~ 35

Author(s):

Peter G. E. Kennedy ◽

Esther Grinfeld ◽

Marie Craigon ◽

Klemens Vierlinger ◽

Douglas Roy ◽

...

Keyword(s):

Gene Expression ◽

Varicella Zoster Virus ◽

Gene Transcription ◽

Spinal Ganglia ◽

Viral Gene ◽

Varicella Zoster Virus Infection ◽

Global Pattern ◽

Varicella Zoster ◽

Viral Gene Transcription ◽

Diagnostic Potential

Varicella-zoster virus (VZV) is a human herpes virus that causes varicella as a primary infection and herpes zoster following reactivation of the virus from a latent state in trigeminal and spinal ganglia. In order to study the global pattern of VZV gene transcription, VZV microarrays using 75-base oligomers to 71 VZV open reading frames (ORFs) were designed and validated. The long-oligonucleotide approach maximizes the stringency of detection and polarity of gene expression. To optimize sensitivity, microarrays were hybridized to target RNA and the extent of hybridization measured using resonance light scattering. Microarray data were normalized to a subset of invariant ranked host-encoded positive-control genes and the data subjected to robust formal statistical analysis. The programme of viral gene expression was determined for VZV (Dumas strain)-infected MeWo cells and SVG cells (an immortalized human astrocyte cell line) 72 h post-infection. Marked quantitative and qualitative differences in the viral transcriptome were observed between the two different cell types using the Dumas laboratory-adapted strain. Oligonucleotide-based VZV arrays have considerable promise as a valuable tool in the analysis of viral gene transcription during both lytic and latent infections, and the observed heterogeneity in the global pattern of viral gene transcription may also have diagnostic potential.

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