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.

FIG. 1.

Transmission electron micrographs of melanoma cells 48 h after infection with ROka (A and C) or 72 h after infection with ROka63D (B and D). A ROka-infected cell contains enveloped virions (A, arrow and arrowheads), while a ROka63D-infected cell contains few virions (B, arrow). A large paracrystalline array of nucleocapsids is seen in the nucleus of a ROka-infected cell (C), while a ROka63D-infected cell shows fewer nucleocapsids (D).

FIG. 2.

VZV ORF63 protein expression by recombinant adenovirus. Human diploid fibroblasts were infected with the replication-defective adenoviruses Ad63 (containing VZV ORF63) or Adblank (containing no insert). Forty-eight hours after infection, cells were harvested and lysates were used for immunoblotting with rabbit polyclonal antibody to the ORF63 protein.

FIG. 3.

(A) Normalization of gE expression levels in VZV-infected cells. Melanoma cells were infected with ROka63D or ROka at the indicated ratios of infected to uninfected cells as described in Materials and Methods. Cells were harvested 48 h postinfection, and lysates were used for immunoblotting with monoclonal antibody to VZV gE. (B) Relative expression levels of each VZV ORF in ROka63D- versus ROka-infected cells. Cells were harvested 48 h postinfection, and mRNA was isolated and used to synthesize radiolabeled cDNA, which was hybridized to VZV arrays. Each point indicates the relative expression level of the respective ORF in ROka63D-infected cells versus ROka-infected cells. Error bars indicate the standard error of the mean. The thick horizontal line represents the mean relative expression level for all ORFs, and the shaded region indicates 1 standard error of the mean. The control targets (left to right) are actin, GAPDH, no DNA, and a negative-control, plasmid-derived sequence (tet). The data represent the average of three independent experiments comprising a total of 10 ROka arrays and 6 ROka63D arrays.

FIG. 4.

Diagram of ORF63 proteins produced by ROka and by ORF63 mutants and their phenotypes in cell culture and in animals. Replication values were determined by dividing the peak titer in mutant-infected cells by the peak titer in ROka-infected cells. Latency values were determined by dividing the percentage of animals latently infected with the mutant by the percentage of animals latently infected with ROka. Data in the replication and latency columns are derived from references and . ORF62 expression values were determined by dividing (ORF62 copy number in mutant-infected cells/ORF62 copy number in ROka-infected cells) by (gB copy number in mutant-infected cells/gB copy number in ROka-infected cells). Data in the ORF62 expression column are derived from RT-PCR. ROka63D encodes a protein in which amino acids 24 to 268 are deleted, and codons 269 to 278 are out of frame. Numbers over the bar corresponding to ROka63-5M indicate sites of alanine substitution for serine or threonine.