Downregulation of Varicella-Zoster Virus (VZV) Immediate-Early ORF62 Transcription by VZV ORF63 Correlates with Virus Replication In Vitro and with Latency

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) protein is expressed during latency in human sensory ganglia. Deletion of ORF63 impairs virus replication in cell culture and establishment of latency in cotton rats. We found that cells infected with a VZV ORF63 deletion mutant yielded low titers of cell-free virus and produced very few enveloped virions detectable by electron microscopy compared with those infected with parental virus. Microarray analysis of cells infected with a recombinant adenovirus expressing ORF63 showed that transcription of few human genes was affected by ORF63; a heat shock 70-kDa protein gene was downregulated, and several histone genes were upregulated. In experiments using VZV transcription arrays, deletion of ORF63 from VZV resulted in a fourfold increase in expression of ORF62, the major viral transcriptional activator. A threefold increase in ORF62 protein was observed in cells infected with the ORF63 deletion mutant compared with those infected with parental virus. Cells infected with ORF63 mutants impaired for replication and latency (J. I. Cohen, T. Krogmann, S. Bontems, C. Sadzot-Delvaux, and L. Pesnicak, J. Virol. 79:5069-5077, 2005) showed an increase in ORF62 transcription compared with those infected with parental virus. In contrast, cells infected with an ORF63 mutant that is not impaired for replication or latency showed ORF62 RNA levels equivalent to those in cells infected with parental virus. The ability of ORF63 to downregulate ORF62 transcription may play an important role in virus replication and latency.

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Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC

Journal of Virology ◽

10.1128/jvi.76.21.11012-11023.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 11012-11023 ◽

Cited By ~ 31

Author(s):

Hitoshi Sato ◽

Lawrence D. Callanan ◽

Lesley Pesnicak ◽

Tammy Krogmann ◽

Jeffrey I. Cohen

Keyword(s):

Varicella Zoster Virus ◽

Deletion Mutant ◽

Host Protein ◽

Parental Virus ◽

Rna Cleavage ◽

Reading Frame ◽

Varicella Zoster ◽

Cotton Rats ◽

Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) open reading frame 17 (ORF17) is homologous to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). HSV vhs induces degradation of mRNA and rapid shutoff of host protein synthesis. An antibody to ORF17 protein detected a 46-kDa protein in VZV-infected cells. While HSV vhs is located in virions, VZV ORF17 protein was not detectable in virions. ORF17 protein induced RNA cleavage, but to a substantially lesser extent than HSV-1 vhs. Expression of ORF17 protein did not inhibit expression from a β-galactosidase reporter plasmid, while HSV type 1 vhs abolished reporter expression. Two VZV ORF17 deletion mutants were constructed to examine the role of ORF17 in virus replication. While the ORF17 VZV mutants grew to peak titers that were similar to those of the parental virus at 33°C, the ORF17 mutants grew to 20- to 35-fold-lower titers than parental virus at 37°C. ORF62 protein was distributed in a different pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37°C compared to 33°C. Inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus. The importance of ORF17 protein for viral replication at 37°C but not at 33°C suggests that this protein may facilitate the growth of virus in certain tissues in vivo.

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Absence or Overexpression of the Varicella-Zoster Virus (VZV) ORF29 Latency-Associated Protein Impairs Late Gene Expression and Reduces VZV Latency in a Rodent Model

Journal of Virology ◽

10.1128/jvi.01220-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1586-1591 ◽

Cited By ~ 20

Author(s):

Jeffrey I. Cohen ◽

Tammy Krogmann ◽

Lesley Pesnicak ◽

Mir A. Ali

Keyword(s):

Varicella Zoster Virus ◽

Deletion Mutant ◽

Latent Infection ◽

Rodent Model ◽

Parental Virus ◽

Late Gene Expression ◽

Varicella Zoster ◽

Reduced Frequency ◽

Immediate Early Proteins ◽

In Cells

ABSTRACT Varicella-zoster virus (VZV) ORF29 encodes the viral single-stranded DNA binding protein and is expressed during latency in human ganglia. We constructed an ORF29 deletion mutant virus and showed that the virus could replicate only in cells expressing ORF29. An ORF29-repaired virus, in which ORF29 was driven by a cytomegalovirus promoter, grew to peak titers similar to those seen with the parental virus. The level of ORF29 protein in cells infected with the repaired virus was greater than that seen with parental virus. Infection of cells with either the ORF29 deletion or repaired virus resulted in similar levels of VZV immediate-early proteins but reduced levels of glycoprotein E compared to those observed with parental virus. Cotton rats infected with the ORF29 deletion mutant had a markedly reduced frequency of latent infection in dorsal root ganglia compared with those infected with parental virus (P < 0.00001). In contrast, infection of animals with the ORF29 deletion mutant resulted in a frequency of ganglionic infection at 3 days similar to that seen with the parental virus. Animals infected with the ORF29-repaired virus, which overexpresses ORF29, also had a reduced frequency of latent infection compared with those infected with parental virus (P = 0.0044). These studies indicate that regulation of ORF29 at appropriate levels is critical for VZV latency in a rodent model.

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

Journal of Virology ◽

10.1128/jvi.78.21.11833-11840.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11833-11840 ◽

Cited By ~ 42

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.

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Varicella-Zoster Virus ORF4 Latency-Associated Protein Is Important for Establishment of Latency

Journal of Virology ◽

10.1128/jvi.79.11.6969-6975.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6969-6975 ◽

Cited By ~ 35

Author(s):

Jeffrey I. Cohen ◽

Tammy Krogmann ◽

Jeffrey P. Ross ◽

Lesley Pesnicak ◽

Elena A. Prikhod'ko

Keyword(s):

Varicella Zoster Virus ◽

Deletion Mutant ◽

Immediate Early ◽

Varicella Zoster ◽

Early Protein ◽

Orf4 Protein ◽

Cotton Rats ◽

In Cells ◽

Hsv 1

ABSTRACT Varicella-zoster virus (VZV) encodes at least six genes that are expressed during latency. One of the genes, ORF4, encodes an immediate-early protein that is present in the virion tegument. ORF4 RNA and protein have been detected in latently infected human ganglia. We have constructed a VZV mutant deleted for ORF4 and have shown that the gene is essential for replication in vitro. The ORF4 mutant virus could be propagated when grown in cells infected with baculovirus expressing the ORF4 protein under the human cytomegalovirus immediate-early promoter. In contrast, the VZV ORF4 deletion mutant could not be complemented in cells expressing herpes simplex virus type 1 (HSV-1) ICP27, the homolog of ORF4. Cells infected with baculovirus expressing ORF4 did not complement an HSV-1 ICP27 deletion mutant. VZV-infected cotton rats have been used as a model for latency; viral DNA and latency-associated transcripts are expressed in dorsal root ganglia 1 month or more after experimental infection. Cotton rats inoculated with VZV lacking ORF4 showed reduced frequency of latency compared to animals infected with the parental or ORF4-rescued virus. Thus, in addition to VZV ORF63, which was previously shown to be critical for efficient establishment of latency, ORF4 is also important for latent infection.

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Varicella-Zoster Virus IE63, a Major Viral Latency Protein, Is Required To Inhibit the Alpha Interferon-Induced Antiviral Response

Journal of Virology ◽

10.1128/jvi.00325-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7844-7851 ◽

Cited By ~ 56

Author(s):

Aruna P. N. Ambagala ◽

Jeffrey I. Cohen

Keyword(s):

Varicella Zoster Virus ◽

Melanoma Cells ◽

Alpha Interferon ◽

Parental Virus ◽

Initiation Factor ◽

Viral Gene ◽

Α Subunit ◽

Varicella Zoster ◽

In Cells ◽

Hsv 1

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is the most abundant transcript expressed during latency in human sensory ganglia. VZV with ORF63 deleted is impaired for replication in melanoma cells and fibroblasts and for latency in rodents. We found that replication of the ORF63 deletion mutant is fully complemented in U2OS cells, which have been shown to complement the growth of herpes simplex virus type 1 (HSV-1) ICP0 mutants. Since HSV-1 ICP0 mutants are hypersensitive to alpha interferon (IFN-α), we examined the effect of IFN-α on VZV replication. Replication of the ORF63 mutant in melanoma cells was severely inhibited in the presence of IFN-α, in contrast to other VZV mutants that were similarly impaired for replication or to parental virus. The VZV ORF63 mutant was not hypersensitive to IFN-γ. IFN-α inhibited viral-gene expression in cells infected with the ORF63 mutant at a posttranscriptional level. Since IFN-α stimulates gene products that can phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF-2α) and inhibit translation, we determined whether cells infected with the ORF63 mutant had increased phosphorylation of eIF-2α compared with cells infected with parental virus. While phosphorylated eIF-2α was undetectable in uninfected cells or cells infected with parental virus, it was present in cells infected with the ORF63 mutant. Conversely, expression of IE63 (encoded by ORF63) in the absence of other viral proteins inhibited phosphorylation of eIF-2α. Since IFN-α has been shown to limit VZV replication in human skin xenografts, the ability of VZV IE63 to block the effects of the cytokine may play a critical role in VZV pathogenesis.

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Resveratrol inhibition of varicella-zoster virus replication in vitro

Antiviral Research ◽

10.1016/j.antiviral.2006.07.004 ◽

2006 ◽

Vol 72 (3) ◽

pp. 171-177 ◽

Cited By ~ 68

Author(s):

John J. Docherty ◽

Thomas J. Sweet ◽

Erin Bailey ◽

Seth A. Faith ◽

Tristan Booth

Keyword(s):

Varicella Zoster Virus ◽

Virus Replication ◽

Varicella Zoster

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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

Journal of Virology ◽

10.1128/jvi.77.20.11180-11185.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 11180-11185 ◽

Cited By ~ 19

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.

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Functions of the ORF9-to-ORF12 Gene Cluster in Varicella-Zoster Virus Replication and in the Pathogenesis of Skin Infection

Journal of Virology ◽

10.1128/jvi.00303-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5825-5834 ◽

Cited By ~ 51

Author(s):

Xibing Che ◽

Mike Reichelt ◽

Marvin H. Sommer ◽

Jaya Rajamani ◽

Leigh Zerboni ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Gene Cluster ◽

Skin Infection ◽

Cultured Cells ◽

Human Tonsil ◽

Reading Frame ◽

Varicella Zoster ◽

The Individual

ABSTRACT The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKAΔ10/11), ORF11 and ORF12 (POKAΔ11/12), or ORF10, ORF11 and ORF12 (POKAΔ10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKAΔ11, POKAΔ10/11, and POKAΔ11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKAΔ10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.

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Nuclear Accumulation of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Is Inhibited by Phosphorylation Mediated by the VZV Open Reading Frame 66 Protein Kinase

Journal of Virology ◽

10.1128/jvi.74.5.2265-2277.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2265-2277 ◽

Cited By ~ 61

Author(s):

Paul R. Kinchington ◽

Karen Fite ◽

Stephanie E. Turse

Keyword(s):

Protein Kinase ◽

Varicella Zoster Virus ◽

Nuclear Localization ◽

Kinase Activity ◽

Open Reading Frame ◽

Nuclear Accumulation ◽

Protein Kinase Activity ◽

Reading Frame ◽

Varicella Zoster ◽

In Cells

ABSTRACT IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

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Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

10.1101/2020.09.11.294280 ◽

2020 ◽

Cited By ~ 1

Author(s):

Werner J. D. Ouwendijk ◽

Daniel P. Depledge ◽

Labchan Rajbhandari ◽

Tihana Lenac Rovis ◽

Stipan Jonjic ◽

...

Keyword(s):

Gene Expression ◽

Varicella Zoster Virus ◽

Viral Gene Expression ◽

Fusion Transcript ◽

Viral Gene ◽

Reading Frame ◽

Varicella Zoster ◽

Rapid Amplification

SummaryVaricella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

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