Viral gene expression in rat trigeminal ganglia following neonatal infection with varicella-zoster virus

The mechanism of the divergent expression of the varicella-zoster virus (VZV) ORF 28 and ORF 29 genes from a common intergenic DNA element, the ORF 28/29 promoter, is of interest based on the observation that both genes are expressed during VZV lytic infection but only the ORF 29 gene is expressed in latently infected neurons. In the work presented here, expression driven by the ORF 28/29 intergenic region was examined. We found that the promoter activity towards the ORF 29 direction is more responsive to activation by the major viral transactivator IE62 than that towards the ORF 28 direction in the context of our experimental system. Analysis of the functional DNA elements involved in IE62 activation of the bidirectional ORF 28/29 regulatory element revealed that in both transfected and VZV-superinfected cells it is a fusion of two unidirectional promoters overlapping an essential USF binding site but with distinct TATA elements. A single TATA element directs expression in the ORF 28 direction, whereas the two TATA elements directing ORF 29 gene expression are alternatively and differentially utilized for transcription initiation. We also identified an Sp1 site localized proximal to the ORF 28 gene which functions as an activator element for expression in both directions. These results indicate that the ORF 28 and ORF 29 genes can be expressed either coordinately or independently and that the observed expression of only the ORF 29 gene during VZV latency may involve neuron-specific cellular factors and/or structural aspects of the latent viral genome.Varicella-zoster virus (VZV) is the causative agent of two human diseases: varicella (chickenpox) and zoster (shingles). Primary infection gives rise to varicella with characteristic skin lesions resulting from lytic infection of the virus in cutaneous epithelial cells. As a member of the neurotropic alphaherpesvirus subfamily, VZV can establish latency in the dorsal root ganglia following primary infection (4). Latent VZV DNA is predominantly localized in the neurons, although some researchers also identified viral sequences in nonneuronal satellite cells (14,22,23,32,37). Data obtained from human ganglia and animal models indicate that during latent infection, a small subset of lytic viral genes is expressed while most of the VZV genome is transcriptionally quiescent. These latency-expressed genes include open reading frames (ORFs) 63, 62, 29, 21, 4, and 66 (2, 7, 8, 11, 12, 20, 24, 33, 35, 66). In the majority of studies conducted, expression of these genes has been detected at the level of RNA. However, expression of all of these genes at the protein level has also been reported (8,12,33,35,66), raising the possibility of a role for one or more of them as trans-acting factors during latency. While the majority of the expressed proteins detected appear to be cytoplasmic rather than nuclear, the possibility of the presence of protein below the levels of detection in the nuclei or a rapid shuttling between the nucleus and cytoplasm cannot be discounted. Because s...

contrasting

confidence: 99%

“…(iii) Is there a cryptic regulatory element within the ORF 29 coding sequence which directs expression of ORF 29 in latently infected cells? The answers to these questions await further experimentation in VZV latency models which are either extant or under development (7,8,24,31,66).…”

Section: Discussionmentioning

confidence: 99%

The DNA Element Controlling Expression of the Varicella-Zoster Virus Open Reading Frame 28 and 29 Genes Consists of Two Divergent Unidirectional Promoters Which Have a Common USF Site

Yang

Hay

Ruyechan

2004

“…Contrary to previous assumptions that ORF 40 transcripts, encoding the major VZV nucleocapsid protein, were highly expressed during lytic virus infection (3,(6)(7)(8)20), our array analysis suggests and Northern blot analysis confirms that ORF 40 transcripts in productively infected cells are low abundance (23-fold less abundant than ORF 9/9A).…”

Section: Discussionmentioning

confidence: 99%

Array Analysis of Viral Gene Transcription during Lytic Infection of Cells in Tissue Culture with Varicella-Zoster Virus

Cohrs¹,

Hurley²,

Gilden

2003

Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, causes childhood chickenpox (varicella), becomes latent in dorsal root and autonomic ganglia, and reactivates decades later to cause shingles (zoster) and other neurologic complications. Although the sequence and configuration of VZV DNA have been determined, relatively little is known about viral gene expression in productively infected cells. This is in part because VZV is highly cell associated, and sufficient titers of cell-free virus for use in synchronizing infection do not develop. PCR-based transcriptional arrays were constructed to simultaneously determine the relative abundance of the Ϸ70 predicted VZV open reading frames (ORFs). Fragments (250 to 600 bp) from the 5 and 3 end of each ORF were PCR amplified and inserted into plasmid vectors. The virus DNA inserts were amplified, quantitated, and spotted onto nylon membranes. Probing the arrays with radiolabeled cDNA synthesized from VZV-infected cells revealed an increase in the magnitude of the expressed VZV genes from days 1 to 3 after low-multiplicity virus infection but little change in their relative abundance. The most abundant VZV transcripts mapped to ORFs 9/9A, 64, 33/33A, and 49, of which only ORF 9 corresponded to a previously identified structural gene. Array analysis also mapped transcripts to three large intergenic regions previously thought to be transcriptionally silent, results subsequently confirmed by Northern blot and reverse transcription-PCR analysis. Array analysis provides a formidable tool to analyze transcription of an important ubiquitous human pathogen. Varicella-zoster virus (VZV) is propagated by cocultivating infected cells with uninfected cells.Because VZV is cell associated and high titers of cell-free virus that can be used to synchronize infection do not develop, analysis of VZV gene transcription in infected cells in tissue culture has been limited. Three reports described global analysis of VZV transcription in cultures harvested at advanced stages of infection; Northern blot analysis of RNA extracted from late-stage VZV-infected cultures identified 58 to 67 discrete transcripts mapping throughout the virus genome (26, 32), while single-stranded DNA probes used in Northern blots revealed the direction of transcription of 57 of the 58 previously identified VZV transcripts and also identified 20 novel virus transcripts (35). Because the RNA in those studies was sampled at the height of cytopathic effect, the transcripts were presumed to reflect VZV structural genes.To better understand the pattern of virus gene transcription during lytic infection, we constructed PCR-based VZV transcriptional arrays to analyze the magnitude of expression and relative abundance of each predicted VZV gene as well as detect transcription for three large intergenic regions of the virus genome. MATERIALS AND METHODSVirus and cells. VZV (strain Ellen) was propagated as described previously (13) by cocultivation of infected and uninfected BSC-1 cells, a continuous cell line derived...

“…In contrast, during VZV latency, multiple genes are expressed. Transcripts from open reading frames (ORFs) 4, 21, 29, 62, 63, and 66 have been demonstrated in trigeminal or dorsal root ganglia latently infected with VZV from humans (9-13, 15, 22, 31) and from rats (2,21,37). ORF63 transcripts are the most abundant VZV mRNAs expressed during latency (12).…”

mentioning

confidence: 99%

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

Sato

Pesnicak

Cohen

2003

Self Cite

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.Primary infection with varicella-zoster virus (VZV) causes chicken pox, and the virus disseminates throughout the body. Infection of the nervous system during primary infection results in establishment of virus latency in sensory ganglia. The same virus can reactivate years later to cause herpes zoster. Although both VZV and herpes simplex virus (HSV) establish latency in sensory neurons, several aspects of latent infection differ for each virus. VZV usually reactivates once, if at all, during the lifetime of immunocompetent persons, whereas HSV often reactivates multiple times. The latency-associated transcripts are the only viral mRNAs abundantly expressed during latency of HSV, and no viral proteins are expressed. In contrast, during VZV latency, multiple genes are expressed. Transcripts from open reading frames (ORFs) 4, 21, 29, 62, 63, and 66 have been demonstrated in trigeminal or dorsal root ganglia latently infected with VZV from humans (9-13, 15, 22, 31) and from rats (2, 21, 37). ORF63 transcripts are the most abundant VZV mRNAs expressed during latency (12). ORF63 protein has also been detected during latency by several laboratories (16,27,29), and other proteins, including the ORF21, ORF29, ORF62, and ORF66 proteins, have been reported in single studies to be expressed during latency (11,27).A number of VZV genes are dispensable in cell culture (8). Several of these genes have important roles in viral pathogenesis. VZV encodes two protein kinases, ORF47 and ORF66 proteins, that are dispensable for virus replication in melanoma cells and fibroblasts (18,19,33). ORF47 protein phosphorylates the VZV ORF62 and ORF63 i...