Varicella-zoster virus gene 21: transcriptional start site and promoter region

Abstract

Varicella-zoster virus (VZV) causes chicken pox (varicella), becomes latent in dorsal root ganglia, and reactivates decades later to cause shingles (zoster). During latency, the entire VZV genome is present in a circular form, from which genes 21, 29, 62, and 63 are transcribed. Immediate-early (IE) VZV genes 62 and 63 encode regulators of virus gene transcription, and VZV gene 29 encodes a major DNA-binding protein. However, little is known about the function of VZV gene 21 or the control of its transcription. Using primer extensions, we mapped the start of VZV gene 21 transcription in VZV-infected cells to a single site located at -79 nucleotides (nt) with respect to the initiation codon. To identify the VZV gene 21 promoter, the 284-bp region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase gene. In transient-transfection assays, the VZV gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV gene 21 promoter. By use of transient-cotransfection assays in conjunction with 5' deletions of the VZV gene 21 promoter, a 40-bp segment was shown to be responsible for the transactivation of the VZV gene 21 promoter by IE62. This region was located at -96 to -56 nt with respect to the 5' start of gene 21 transcription.

FIG. 1

Northern blot analysis of VZV-infected BSC-1 RNA. (A) Total RNA (20 μg) extracted from control BSC-1 cells (lanes C) and VZV-infected BSC-1 cells (lanes V) along with RNA standards (lane M; Gibco-BRL) was resolved in 1% agarose gels containing 0.5 mM methylmercury(II) hydroxide and stained with 0.5 μg of ethidium bromide per ml in 0.5 M ammonium acetate. (B and C) RNA was transferred to a nylon-based membrane and probed for VZV gene 21 transcripts (B) or β-actin transcripts (C). VZV gene 21 transcripts are visible as a discrete 3.1-kb band in VZV-infected cell RNA. Both control and VZV-infected cell RNA contain discrete 1.8-kb β-actin transcripts.

FIG. 2

Location of the 5′ start of RNA transcription for VZV gene 21. Total RNA (5 μg) from either VZV-infected (lanes V) or uninfected (lanes C) BSC-1 cells was annealed to oligonucleotide primer 21pel, 21pe2, or 21pe3 end labeled with ³²P (Table 1). First-strand cDNA was synthesized, and the extended product was resolved by gel electrophoresis. The DNA sequence of the SalI C fragment of VZV DNA primed with the respective oligonucleotides was used to size the cDNA products. The entire gel image as well as an enlargement of the region containing extended products is shown. With all three primers, the cDNA product obtained from VZV-infected cell RNA (closed arrows in lanes V) terminated at the identical adenosine located at nt 30681 on the VZV genome. Minor extended products obtained from VZV-infected cell RNA (open arrows in lanes V) were also observed. No product was observed when uninfected cell RNA was used in the cDNA synthesis reaction (lanes C).

FIG. 3

Schematic representation of the VZV DNA between ORFs 20 and 21. The VZV genome consists of unique long (U_L) and unique short (U_S) segments of DNA, each bounded by inverted and repeated DNA sequences (TR_L/IR_L and IR_S/TR_S). ORFs 20 and 21 are oriented in opposite directions, and both map within the SalI C fragment (positions 23454 to 35936) within the U_L. The 284-bp DNA segment separating ORFs 20 and 21 contains one potential IE62 binding site and three TATAA-like boxes. The 5′ start site of gene 21 transcription is located at nt 30681, and the 3′ end of the transcript has been mapped to nt 33888 and nt 33895 (7, 10). The boundary of the CAT reporter constructs used to locate the VZV gene 21 promoter are shown.

FIG. 4

The VZV gene 21 promoter is silent in uninfected cells. The 284-bp VZV DNA segment separating ORFs 20 and 21 was inserted into the CAT reporter plasmid and used to transfect either uninfected cells (lane 2) or VZV-infected cells (lane 4). Controls included the CAT reporter plasmid lacking a promoter transfected into uninfected cells (lane 1) or VZV-infected cells (lane 3) and a CMV IE promoter driving CAT transfected into uninfected cells (lane 5). CAT assays were performed in duplicate, and the average acetylation of chloramphenicol (%CAT) showed that the VZV gene 21 promoter does not function in uninfected cells (−VZV) but is active in VZV-infected cells (+VZV). The amount of promoter activity in infected cells above that in uninfected cells (fold) showed that gene 21 promoter activity was approximately 650-fold higher in VZV-infected cells than in uninfected cells.

FIG. 5

The VZV gene 21 promoter is transactivated by IE62. CAT reporter plasmids, either promoterless (lane 1) or containing the 284-bp VZV ORF 20-ORF 21 intergenic region (lanes 2 to 7) or the CMV IE promoter (lane 8), were transfected into cells either alone (lanes 1, 2, and 8) or with plasmids expressing various VZV transactivators (ORFs 4, 10, 61, 62, and 63) (lanes 3 to 7). Duplicate CAT assays indicated that the VZV gene 21 promoter is transactivated by VZV IE62 but not by the proteins encoded by VZV genes 4, 10, 61, and 63. Transactivation of VZV gene 21 promoter by IE62 is ∼42-fold higher than VZV gene 21 promoter activity in the absence of IE62. %CAT, average percent chloramphenicol acetylation; sd, standard deviation.

FIG. 6

5′ boundary of the gene 21 promoter. Duplicate CAT assays were performed on extracts of uninfected cells that had been transfected with CAT reporter constructs containing either the entire 284-bp DNA segment separating ORF 20 and ORF 21 (p21), a 496-bp extension (p21Z), or various 5′ truncation mutations (p21A, p21B, p21C, and p21Δ) in the presence of the IE62 expression plasmid. The 5′ boundary of the gene 21 promoter was located to a region between nt 30585 (p21A) and nt 30626 (p21B). %CAT, average percent chloramphenicol acetylation; sd, standard deviation.