scholarly journals The Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Is Critical for Establishment of Latency

2004 ◽  
Vol 78 (21) ◽  
pp. 11833-11840 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Edward Cox ◽  
Lesley Pesnicak ◽  
Shamala Srinivas ◽  
Tammy Krogmann
Keyword(s):  
Varicella Zoster Virus ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Mutant Virus ◽  
Open Reading Frame ◽  
Parental Virus ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Latently Infected

ABSTRACT Varicella-zoster virus (VZV) expresses at least six viral transcripts during latency. One of these transcripts, derived from open reading frame 63 (ORF63), is one of the most abundant viral RNAs expressed during latency. The VZV ORF63 protein has been detected in human and experimentally infected rodent ganglia by several laboratories. We have deleted >90% of both copies of the ORF63 gene from the VZV genome. Animals inoculated with the ORF63 mutant virus had lower mean copy numbers of latent VZV genomes in the dorsal root ganglia 5 to 6 weeks after infection than animals inoculated with parental or rescued virus, and the frequency of latently infected animals was significantly lower in animals infected with the ORF63 mutant virus than in animals inoculated with parental or rescued virus. In contrast, the frequency of animals latently infected with viral mutants in other genes that are equally or more impaired for replication in vitro, compared with the ORF63 mutant, is similar to that of animals latently infected with parental VZV. Examination of dorsal root ganglia 3 days after infection showed high levels of VZV DNA in animals infected with either ORF63 mutant or parental virus; however, by days 6 and 10 after infection, the level of viral DNA in animals infected with the ORF63 mutant was significantly lower than that in animals infected with parental virus. Thus, ORF63 is not required for VZV to enter ganglia but is the first VZV gene shown to be critical for establishment of latency. Since the present vaccine can reactivate and cause shingles, a VZV vaccine based on the ORF63 mutant virus might be safer.

scholarly journals Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

2006 ◽  
Vol 80 (4) ◽  
pp. 1710-1723 ◽  
Author(s):  
Amie J. Eisfeld ◽  
Stephanie E. Turse ◽  
Sara A. Jackson ◽  
Edwina C. Lerner ◽  
Paul R. Kinchington
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Nuclear Import ◽  
Regulatory Protein ◽  
Recombinant Baculovirus ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Transcriptional Regulatory

ABSTRACT IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

scholarly journals Latency In Vitro of Varicella-Zoster Virus in Cells Derived from Human Fetal Dorsal Root Ganglia

1992 ◽  
Vol 32 (6) ◽  
pp. 699-703 ◽  
Author(s):  
Eli Somekh ◽  
Davol G Tedder ◽  
Abbas Vafai ◽  
José G Assouline ◽  
Stephen E Straus ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Varicella Zoster ◽  
In Cells

scholarly journals Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency

2005 ◽  
Vol 79 (8) ◽  
pp. 5069-5077 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Tammy Krogmann ◽  
Sebastien Bontems ◽  
Catherine Sadzot-Delvaux ◽  
Lesley Pesnicak
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

scholarly journals Varicella-Zoster Virus Open Reading Frame 10 Is a Virulence Determinant in Skin Cells but Not in T Cells In Vivo

2006 ◽  
Vol 80 (7) ◽  
pp. 3238-3248 ◽  
Author(s):  
Xibing Che ◽  
Leigh Zerboni ◽  
Marvin H. Sommer ◽  
Ann M. Arvin
Keyword(s):  
Varicella Zoster Virus ◽  
Open Reading Frame ◽  
Early Gene ◽  
Pathogenic Potential ◽  
Reading Frame ◽  
Virion Assembly ◽  
Varicella Zoster ◽  
Skin Cells

ABSTRACT The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKAΔ10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKAΔ10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKAΔ10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.

scholarly journals Dissection of a Novel Nuclear Localization Signal in Open Reading Frame 29 of Varicella-Zoster Virus

2005 ◽  
Vol 79 (20) ◽  
pp. 13070-13081 ◽  
Author(s):  
Christina L. Stallings ◽  
Saul Silverstein
Keyword(s):  
Dorsal Root Ganglia ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Dorsal Root ◽  
Open Reading Frame ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Localization Signal

ABSTRACT Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin α- and β-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin α fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin α. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.

scholarly journals Varicella-Zoster Virus Open Reading Frame 21, Which Is Expressed during Latency, Is Essential for Virus Replication but Dispensable for Establishment of Latency

2003 ◽  
Vol 77 (2) ◽  
pp. 1211-1218 ◽  
Author(s):  
Dongxiang Xia ◽  
Shamala Srinivas ◽  
Hitoshi Sato ◽  
Lesley Pesnicak ◽  
Stephen E. Straus ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Deletion Mutant ◽  
Latent Infection ◽  
Open Reading Frame ◽  
Protein Insertion ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Varicella-zoster virus (VZV) open reading frame 21 (ORF21) is one of at least five VZV genes expressed in latently infected human and rodent ganglia. To determine whether ORF21 is required for latent and lytic infection, we deleted 99% of ORF21 from the viral genome. The ORF21 deletion mutant virus could be propagated only in a cell line expressing the ORF21 protein. Insertion of the herpes simplex virus type 1 (HSV-1) homolog of VZV ORF21, HSV-1 UL37, into the ORF21 deletion mutant failed to complement the mutant for growth in cell culture. Inoculation of cotton rats with the ORF21 deletion virus resulted in latent infection in numbers of animals similar to those infected after inoculation with the parental virus. The mean numbers of latent VZV genomes were similar in animals infected with parental and ORF21 deletion viruses. Transcription of ORF63, another latency-associated gene, was detected in ganglia from similar numbers of animals infected with the mutant and parental viruses. Thus, ORF21 is the first VZV gene expressed during latency that has been shown to be dispensable for the establishment of latent infection.

scholarly journals Varicella-Zoster Virus Open Reading Frame 2 Encodes a Membrane Phosphoprotein That Is Dispensable for Viral Replication and for Establishment of Latency

2002 ◽  
Vol 76 (7) ◽  
pp. 3575-3578 ◽  
Author(s):  
Hitoshi Sato ◽  
Lesley Pesnicak ◽  
Jeffrey I. Cohen
Keyword(s):  
Varicella Zoster Virus ◽  
Deletion Mutant ◽  
Latent Infection ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Herpesvirus Type ◽  
Varicella Zoster ◽  
Latently Infected ◽  
Simplex Virus ◽  
Open Reading Frame 2

ABSTRACT Varicella-zoster virus (VZV) encodes six genes that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 2 (ORF2), was expressed as a 31-kDa phosphoprotein in the membranes of infected cells. Unlike equine and bovine herpesvirus type 1 ORF2 homologs that are associated with virions, VZV virions contained no detectable ORF2 protein. The ORF2 deletion mutant established a latent infection in cotton rats at a frequency and with a number of VZV genomes similar to that of the parental virus. ORF63 transcripts, a hallmark of latent infection, were present in ganglia latently infected with both the ORF2 deletion mutant and parental VZV. Thus, ORF2 is the first VZV gene shown to be dispensable for establishment of latent infection in an animal model.

scholarly journals A unique spliced varicella-zoster virus latency transcript represses expression of the viral transactivator gene 61

2017 ◽  
Author(s):  
Daniel P. Depledge ◽  
Werner J. D. Ouwendijk ◽  
Tomohiko Sadaoka ◽  
Shirley E. Braspenning ◽  
Yasuko Mori ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Functional Characterization ◽  
Skin Lesions ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Viral Transactivator ◽  
Virus Latency ◽  
Latently Infected ◽  
Alternatively Spliced

During primary infection, neurotropic alphaherpesviruses (αHVs) gain access to neurons in sensory and cranial ganglia establishing lifelong latent infection from which they can later reactivate to cause debilitating disease1. For most αHVs, including the best-studied herpes simplex type 1 ( HSV-1), viral latency is characterized by expression of a single or restricted set of transcripts that map antisense to the open reading frame (ORF) homologous to the major HSV immediate early viral transactivator, ICP02. These latency transcripts, either directly or through encoded miRNAs or proteins, repress expression of the ICP0 orthologues3–5. The exception is varicella-zoster virus (VZV), an αHV which infects over 90% of adults and for which neither a canonical latency transcript1,6–8 nor a putative mechanism for repressing lytic transcription during latency have been identified. Here, we describe the discovery and functional characterization of a VZV latency transcript (VLT), that maps antisense to VZV ORF 61 (the VZV ICP0 homologue9,10), and which is consistently expressed in neurons of latently infected human trigeminal ganglia (TG). VLT encodes a protein with late kinetics during lytic VZV infection in vitro and in zoster skin lesions. Whereas multiple alternatively spliced VLT isoforms are expressed during lytic VZV infection, a single unique VLT isoform that specifically suppresses ORF61 gene expression predominates in latently VZV-infected human TG. The discovery of VLT directly unifies the latent VZV transcription program with those of better-characterized αHVs, removing longstanding barriers to understanding VZV latency and paving the way for research into the development of vaccines that do not establish latency or reactivate, and drugs that eradicate latent VZV.

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