scholarly journals Characterization of Varicella-Zoster Virus Gene 21 and 29 Proteins in Infected Cells

2002 ◽  
Vol 76 (14) ◽  
pp. 7228-7238 ◽  
Author(s):  
Randall J. Cohrs ◽  
Jeanne Wischer ◽  
Carrie Essman ◽  
Donald H. Gilden
Keyword(s):  
Varicella Zoster Virus ◽  
Virus Genome ◽  
Open Reading Frames ◽  
Productive Infection ◽  
Gene Promoters ◽  
Varicella Zoster ◽  
Latently Infected ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Varicella-zoster virus (VZV) transcription is limited in latently infected human ganglia. Note that much of the transcriptional capacity of the virus genome has not been analyzed in detail; to date, only VZV genes mapping to open reading frames (ORFs) 4, 21, 29, 62, and 63 have been detected. ORF 62 encodes the major immediate-early virus transcription transactivator IE62, ORF 29 encodes the major virus DNA binding protein, and ORF 21 encodes a protein associated with the developing virus nucleocapsid. We analyzed the cellular location of proteins encoded by ORF 21 (21p) and ORF 29 (29p), their phosphorylation state during productive infection, and their ability form a protein-protein complex. The locations of both 21p and 29p within infected cells mimic those of their herpes simplex virus type 1 (HSV-1) homologues (UL37 and ICP8); however, unlike these homologues, 21p is not phosphorylated and neither 21p nor 29p exhibits a protein-protein interaction. Transient transfection assays to determine the effect of 21p and 29p on transcription from VZV gene 20, 21, 28, and 29 promoters revealed no significant activation of transcription by 21p or 29p from any of the VZV gene promoters tested, and 21p did not significantly modulate the ability of IE62 to activate gene transcription. A modest increase in IE62-induced activation of gene 28 and 29 promoters was seen in the presence of 29p; however, IE62-induced activation of gene 28 and 29 promoters was reduced in the presence of 21p. A Saccharomyces cerevisiae two-hybrid analysis of 21p indicated that the protein can activate transcription when tethered within a responsive promoter. Together, the data reveal that while VZV gene 21 and HSV-1 UL37 share homology at the nucleic acid level, these proteins differ functionally.

scholarly journals Analysis of Human Alphaherpesvirus MicroRNA Expression in Latently Infected Human Trigeminal Ganglia

2009 ◽  
Vol 83 (20) ◽  
pp. 10677-10683 ◽  
Author(s):  
Jennifer L. Umbach ◽  
Maria A. Nagel ◽  
Randall J. Cohrs ◽  
Donald H. Gilden ◽  
Bryan R. Cullen
Keyword(s):  
Trigeminal Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Viral Mirnas ◽  
Varicella Zoster ◽  
Wide Range ◽  
Latently Infected ◽  
Infected Cells ◽  
Virally Encoded ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Analysis of cells infected by a wide range of herpesviruses has identified numerous virally encoded microRNAs (miRNAs), and several reports suggest that these viral miRNAs are likely to play key roles in several aspects of the herpesvirus life cycle. Here we report the first analysis of human ganglia for the presence of virally encoded miRNAs. Deep sequencing of human trigeminal ganglia latently infected with two pathogenic alphaherpesviruses, herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), confirmed the expression of five HSV-1 miRNAs, miR-H2 through miR-H6, which had previously been observed in mice latently infected with HSV-1. In addition, two novel HSV-1 miRNAs, termed miR-H7 and miR-H8, were also identified. Like four of the previously reported HSV-1 miRNAs, miR-H7 and miR-H8 are encoded within the second exon of the HSV-1 latency-associated transcript. Although VZV genomic DNA was readily detectable in the three human trigeminal ganglia analyzed, we failed to detect any VZV miRNAs, suggesting that VZV, unlike other herpesviruses examined so far, may not express viral miRNAs in latently infected cells.

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 Comparison of Virus Transcription during Lytic Infection of the Oka Parental and Vaccine Strains of Varicella-Zoster Virus

2006 ◽  
Vol 80 (5) ◽  
pp. 2076-2082 ◽  
Author(s):  
Randall J. Cohrs ◽  
Donald H. Gilden ◽  
Yasuyuki Gomi ◽  
Koichi Yamanishi ◽  
Jeffrey I. Cohen
Keyword(s):  
Varicella Zoster Virus ◽  
Vaccine Virus ◽  
Open Reading Frames ◽  
Gene Promoters ◽  
Virus Reactivation ◽  
Dot Blot ◽  
Global Pattern ◽  
Varicella Zoster ◽  
Infected Cells ◽  
Disproportionate Number

ABSTRACT The attenuated Oka vaccine (V-Oka) strain of varicella-zoster virus (VZV) effectively reduces disease produced by primary infection and virus reactivation. V-Oka was developed by propagation of the Oka parental (P-Oka) strain of VZV in guinea pig and human embryo fibroblasts. Complete DNA sequencing of both viruses has revealed 63 sites that differ between P-Oka and V-Oka, 37 of which are located within 21 unique open reading frames (ORFs). Of the ORFs that differ, ORF 62 contains the greatest number (10) of mutated sites. ORF 62 encodes IE 62, the major immediate-early transactivator of virus genes, and is essential for lytic virus replication. To determine whether a disproportionate number of mutations in ORF 62 might account for virus attenuation, we compared the global pattern of V-Oka gene expression to that of P-Oka. Transcription of ORFs 62, 65, 66, and 67 was suppressed, whereas ORF 41 was elevated in V-Oka-infected cells compared to P-Oka-infected cells (P < 0.01; z test). Suppression of ORF 62, 65, and 66 transcription was confirmed by quantitative dot blot and Western blot analyses. Transient-transfection assays to determine whether mutations within V-Oka-derived IE 62 affected its ability to transactivate VZV gene promoters revealed similar IE 62 transactivation of VZV gene 20, 21, 28, 29, 65, and 66 promoters in both P-Oka and V-Oka. Together, our results indicate that mutations in V-Oka IE 62 alone are unlikely to account for vaccine virus attenuation.

scholarly journals Potential Role for Luman, the Cellular Homologue of Herpes Simplex Virus VP16 (α Gene trans-Inducing Factor), in Herpesvirus Latency

2000 ◽  
Vol 74 (2) ◽  
pp. 934-943 ◽  
Author(s):  
Rui Lu ◽  
Vikram Misra
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Productive Infection ◽  
Trigeminal Ganglia ◽  
Mutant Form ◽  
Gene Promoters ◽  
Intracellular Location ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The cascade of herpes simplex virus (HSV) gene expression that results in viral replication begins with the activation of viral immediate-early (IE) genes by the virion-associated protein VP16. VP16 on its own is inefficient at associating with complexes formed on IE gene promoters and depends upon the cellular factor HCF for its activity. In this respect VP16 mimics the host basic leucine zipper (bZIP) protein Luman, which also requires HCF for activating transcription. Our objective is to explore interactions between Luman and HCF and to determine if they play a role in the biology of herpesviruses. In this report we show that in cultured cells ectopically expressed Luman was retained in the cytoplasm, where it colocalized with Calnexin, a protein normally associated with the endoplasmic reticulum (ER). Retention of Luman in the ER depends on a hydrophobic segment of the protein that probably serves as a transmembrane domain. Deletion of this domain changed the intracellular location of Luman so that most of the mutant protein was in the nucleus of cells. While HCF was present in the nucleus of most cells, in cells expressing Luman it was retained in the cytoplasm where the two proteins colocalized. This cytoplasmic association of Luman and HCF could also be demonstrated in neurons in trigeminal ganglia removed from cattle soon after death. Cells in tissue culture that expressed Luman, but not a mutant form of the protein that fails to bind HCF, were resistant to a productive infection with HSV type 1 (HSV-1). We hypothesize that similar Luman-HCF interactions in sensory neurons in trigeminal ganglia result in the suppression of viral replication and the establishment of latency. Interestingly, Luman could activate the promoters of IE110 and LAT, two genes that are critical for reactivation of HSV-1 from latency. This suggests a role for Luman in the reactivation process as well.

scholarly journals Epigenetic Regulation of Varicella-Zoster Virus Open Reading Frames 62 and 63 in Latently Infected Human Trigeminal Ganglia

2006 ◽  
Vol 80 (10) ◽  
pp. 4921-4926 ◽  
Author(s):  
Lee Gary ◽  
Donald H. Gilden ◽  
Randall J. Cohrs
Keyword(s):  
Varicella Zoster Virus ◽  
Chromatin Immunoprecipitation ◽  
Latent Infection ◽  
Open Reading Frames ◽  
Trigeminal Ganglia ◽  
Varicella Zoster ◽  
Glycoprotein C ◽  
Latently Infected ◽  
Human Chromosome 4 ◽  
Reading Frames

ABSTRACT Open reading frames (ORFs) 21, 29, 62, 63, and 66 of varicella-zoster virus (VZV) are transcribed during latency in human ganglia. ORF 63 is the most frequently expressed gene, and ORF 62 encodes a transcriptional activator. The mechanisms regulating the expression of these genes are not well understood, although analyses of other alphaherpesviruses indicate a role for chromatin in virus gene regulation during latent infection. Using chromatin immunoprecipitation (ChIP) assays to analyze the euchromatic state of ORFs 62 and 63 compared to the centromere from human chromosome 4 (heterochromatic) and the human glyceraldehyde-3-phosphate dehydrogenase promoter (euchromatic), we show that the promoters of ORFs 62 and 63 are associated with the histone protein H3K9(Ac) and thus maintained in a euchromatic state during latency. Conversely, the promoters of ORF 36 (thymidine kinase) and ORF 14 (glycoprotein C), genes expressed during lytic but not latent infection, were not enriched in the fraction of latently infected ganglia that bound to anti-H3K9(Ac) antibody. A ChIP assay using productively infected MeWo cells revealed that VZV ORFs 62, 63, 36, and 14 are all euchromatic. Together, these data indicate that the expression of the two latency-related VZV genes, ORFs 62 and 63, is regulated epigenetically through chromatin structure.

scholarly journals Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

2016 ◽  
Vol 90 (19) ◽  
pp. 8673-8685 ◽  
Author(s):  
Erin M. Buckingham ◽  
Keith W. Jarosinski ◽  
Wallen Jackson ◽  
John E. Carpenter ◽  
Charles Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Endocytic Pathway ◽  
Autophagic Flux ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Cytoplasmic Vesicles ◽  
Infected Cells ◽  
Autophagy Pathway ◽  
Simplex Virus ◽  
2D And 3D

ABSTRACTVaricella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCEVZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

scholarly journals Quantitation of Latent Varicella-Zoster Virus and Herpes Simplex Virus Genomes in Human Trigeminal Ganglia

1999 ◽  
Vol 73 (12) ◽  
pp. 10514-10518 ◽  
Author(s):  
Stephanie R. Pevenstein ◽  
Richard K. Williams ◽  
Daniel McChesney ◽  
Erik K. Mont ◽  
John E. Smialek ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Ganglion Cells ◽  
Trigeminal Ganglia ◽  
Varicella Zoster ◽  
Glycoprotein G ◽  
Simplex Virus ◽  
Virus Genomes ◽  
Hsv 1

ABSTRACT Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 ± 1,082 (standard error of the mean) or 109 genomes/105 cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 ± 38 genomes/105 ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.

scholarly journals Frequency and Abundance of Alphaherpesvirus DNA in Human Thoracic Sympathetic Ganglia

2014 ◽  
Vol 88 (14) ◽  
pp. 8189-8192 ◽  
Author(s):  
Maria A. Nagel ◽  
April Rempel ◽  
Jonathon Huntington ◽  
Forrest Kim ◽  
Alexander Choe ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Sympathetic Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Varicella Zoster ◽  
Target Organs ◽  
Simplex Virus ◽  
Visceral Disease ◽  
Hsv 1

Alphaherpesvirus reactivation from thoracic sympathetic ganglia (TSG) and transaxonal spread to target organs cause human visceral disease. Yet alphaherpesvirus latency in TSG has not been well characterized. In this study, quantitative PCR detected varicella-zoster virus (VZV), herpes simplex virus 1 (HSV-1), and HSV-2 DNA in 117 fresh TSG obtained postmortem from 15 subjects. VZV DNA was found in 76 (65%) ganglia from all subjects, HSV-1 DNA was found in 5 (4%) ganglia from 3 subjects, and no HSV-2 was found.

scholarly journals A Functional YNKI Motif in the Short Cytoplasmic Tail of Varicella-Zoster Virus Glycoprotein gH Mediates Clathrin-Dependent and Antibody-Independent Endocytosis

2003 ◽  
Vol 77 (7) ◽  
pp. 4191-4204 ◽  
Author(s):  
Tracy Jo Pasieka ◽  
Lucie Maresova ◽  
Charles Grose
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Cytoplasmic Tail ◽  
Dependent Manner ◽  
Independent Manner ◽  
Varicella Zoster ◽  
Cytoplasmic Tails ◽  
Infected Cells ◽  
Simplex Virus ◽  
Alignment Analysis

ABSTRACT The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.

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