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

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Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus.Primary VZV infection begins with inoculation of the respiratory mucosa, followed by cell-associated viremia and the rash of chickenpox. During primary infection, the virus establishes latency in sensory ganglia and can subsequently reactivate to cause shingles. At least six VZV open reading frames (ORFs), ORF4, -21, -29, -62, -63, and -66, are expressed during latency (11-13, 29, 38). ORF63 is the most prevalent and abundant VZV transcript expressed in latently infected human ganglia (11,13).VZV ORF63 encodes a 278-amino-acid protein, which is located in the tegument of virions (32) and is expressed as an immediate-early (IE) protein (14). IE63 is extensively modified in infected cells as a 45-kDa protein (14) that is phosphorylated by the VZV ORF47 kinase (30) and cellular casein kinases (5). IE63 binds to IE62, the major transactivator protein of VZV, and up-regulates expression of the VZV glycoprotein I (gI) promoter (36). IE63 has been reported to act as a transcriptional repressor for a number of VZV and heterologous viral and cellular promoters (5, 17). IE63 promotes the survival of cultured primary human neuronal cells (22). Point mutations in phosphorylation sites prior to the last 70 amino acids of IE63 result in mutant viruses impaired for replication (3,8) and for establishment of latent infection in rodents (8). During lytic replication in vitro, IE63 is located predominantly in the nucleus; however, during latency, the protein is detected in the cytoplasm of sensory neurons (18,35,37).The innate immune response is critical for defense against invading viruses. In response to virus infection, a number of signal transduction pathways are activated, including activation of genes that encode type I interferons (IFNs), such as IFN-␣ and IFN-␤. These IFNs inhibit VZV replication in vitro (4, 15). VZV infection of human skin xenografts in SCID mice results in downregulation of IFN-␣ in infected cells and increased expression of IFN-␣ in the surrounding uninfected epidermal cells, which may delay the appearance of skin lesions (33, 34). These findings indicate that IFN-␣ plays an important role in VZV pathogenesis.Previously, we reported the generation of a recombinant VZV in which over 90% of both copies of ORF63 were deleted (7). The ORF63 deletion mutant was impaired for replication in human melanoma cells and for establishment of latency in rodents. Here, we report that the ORF63 deletion mutant is hypersensitive to human IFN-␣ compared to parental virus or other VZV deletion mutants that are impaired for replication in human melanoma cells. IFN-␣ confers cellular resistance against virus infection by at least two cellular proteins, doublestranded RNA-activated protein kinase (PKR) and 2Ј-5Јoligoad-enylate synthetase. Activated PKR phosphorylates the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣), thereby preventing initiation of translation. We demonstrate that cells infected with the VZV ORF63 deletion mutant have increased levels o...

Section: Discussionmentioning

confidence: 99%

“…VZV ROka63D, ROka61D, and ROka67D have ORF63, -61, and -67 deleted (7,9,10). Recombinant adenovirus Ad63 expressing VZV IE63 and green fluorescent protein and recombinant adenovirus Adblank expressing green fluorescent protein alone were described previously (23).…”

Section: Cells and Virusesmentioning

confidence: 99%

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

Ambagala

Cohen

2007

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“…Mutational analysis has identified two nuclear localization signals (NLSs) within the C terminus that are required for nuclear localization (2, 5). ORF63p is a transcriptional repressor and activator of cellular and viral promoters (5,22,24,30,31,35,42,47,68). It carries out these functions by interacting with ORF62p (the major VZV transactivator protein), RNA polymerase II, and the host transcriptional machinery (2, 24, 47, 58).…”

mentioning

confidence: 99%

Nuclear Import of the Varicella-Zoster Virus Latency-Associated Protein ORF63 in Primary Neurons Requires Expression of the Lytic Protein ORF61 and Occurs in a Proteasome-Dependent Manner

Walters

Kyratsous²,

Wan

et al. 2008

Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shownto be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, is the etiological agent of chicken pox (varicella) and shingles (zoster). Upon primary infection of humans, VZV invades the dermis and epidermis, resulting in chicken pox. Following primary infection, the virus infects cranial nerve and dorsal root ganglia to form a latent infection. VZV can subsequently reactivate to cause shingles (28). During latency, only a subset of VZV genes is expressed. 19,37,49) and proteins (15,29,37,46,48) from open reading frames (ORFs) 4, 21, 29, 62, 63, and 66 have been detected in latently infected human ganglia. These latency-associated proteins (LAPs) appear to accumulate only in the cytoplasm of latently infected neurons. This is in contrast to their nuclear and cytoplasmic localizations during lytic and reactivated VZV infection (29,46,48). These data suggest a correlation between the nuclear import of LAPs and the outcome of VZV infection. Of all the transcripts detected in latently infected human ganglia, ORF63 is the most prevalent and abundant (13, 16). In addition, ORF63 transcripts and protein were detected in the ganglia of experimentally infected rodents (8, 21, 38). Therefore, ORF63 transcription and accumulation of the protein in the cytoplasm of latently infected neurons are some of the hallmarks of VZV latency.VZV ORF63 encodes a 278-amino-acid protein (ORF63p) (20) that is expressed as an immediate-early protein and is present in the viral tegument (41). The protein is extensively phosphorylated during transient expression and lytic VZV infection in cell culture by both host and viral kinases (2,5,18,30,33,39,40,61). Mutational analysis...

“…8A and B), only empty capsids could be observed. Examination of numerous ⌬54S sections revealed the accumulation of empty capsids in groups or clusters which represent crystalline packing of large numbers of capsids, as reported by others (47)(48)(49)(50)(51).…”

Section: Isolation Of An Orf54-expressing Cell Line Arpe54mentioning

confidence: 76%

“…ARPE19 cells were infected with approximately 100 PFU of VZV-infected cell stock (VZV LUC , VZV54 48 r , or VZV54 407 r ) per well. Sensitivity of isolates was determined by diluting the compound to the desired concentrations (0.03, 0.06, 0.125, 0.250, 0.500, 1.00, or 2.00 g/ml) in MEM containing 0.3% dimethyl sulfoxide (DMSO) at the time of infection.…”

Section: Methodsmentioning

confidence: 99%

The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

Visalli

House

Selariu

et al. 2014

The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, ⌬54L (full ORF deletion) and ⌬54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, ⌬54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in ⌬54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (U L ) and a unique short region (U S ) flanked by inverted repeats. DNA from cells infected with parental VZV (VZV LUC strain) contained the predicted U L and U S termini, whereas cells infected with ⌬54S contained neither. This result demonstrates that ⌬54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs ⌬54L and ⌬54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. IMPORTANCE Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections.Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-␣-methylbenzyl-N=-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.H erpesvirus DNA encapsidation is understood only in general terms. The mechanistic events and the function(s) of the proteins involved are still under investigation. During viral replication, progeny DNA genomes are synthesized in the nucleus as long, branched head-to-tail concatemers. The capsid proteins are synt...