The varicella-zoster virus open reading frame 63 latency-associated protein is critical for establishment of latency

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.

FIG. 1.

Construction of recombinant VZV with a deletion in ORF63 and ORF70. The VZV genome is 124,884 bp long (top line) and contains unique long (UL), unique short (US), internal repeat (IR), and terminal repeat (TR) regions. The four cosmids used to produce infectious virus (cosmids VZV NotIA, NotIBD, MstIIB, and MstIIA) span the VZV genome are shown in the middle of the figure. In cosmid MstIIA-63D, codons 24 to 268 of ORF63 and ORF70 (with a frameshift after codon 268) were deleted.

FIG. 2.

Southern blots of virion DNA from cells infected with parental virus (ROka) or viruses in which ORF63 was deleted. ROka, ROka63DA, and ROka63DB DNAs (A) or ROka and ROka63DAR DNAs (B) were digested with BamHI and hybridized to an ORF63 probe. The positions of markers showing the size of the DNA (in kilobase pairs) are indicated at the sides of the gels.

FIG. 3.

Immunoblot of ORF63 expression in cells infected with recombinant VZV. A 45-kDa protein that reacts with antibody to ORF63 protein (α63) is present in cells infected with ROka and ROka63DAR, but not in cells infected with ROka63DA or ROka63DB (A and C). Bands of 70 to 90 kDa that react with antibody to VZV gE (αgE) are present in cells infected with VZV ROka, ROka63DA, and ROka63DB (B). Equivalent amounts of cell lysates were loaded for the cells infected with VZV mutants in which ORF63 had been deleted in panels A and B, but larger amounts of cell lysates were loaded for the cells infected with VZV mutants in which ORF63 had been deleted than for the cells infected with the parental virus. The positions of markers showing the sizes of proteins (in kilodaltons) are indicated to the right of the gels.

FIG. 4.

Growth in melanoma cells of parental VZV and mutant viruses in which ORF63 had been deleted. Cells were infected with the indicated viruses. Each day after infection, the cells were treated with trypsin, and the virus titer was determined.

FIG. 5.

Expression of VZV thymidine kinase (A) and glycoprotein E (B) proteins in cells infected with parental VZV or virus in which ORF63 had been deleted. Cell lysates were immunoprecipitated with antibody to thymidine kinase (αTK) or to glycoprotein E (αgE). The positions of VZV thymidine kinase (35 kDa) and glycoprotein E (gE) (95 kDa) are indicated by the arrowheads. The positions of markers showing the sizes of proteins (in kilodaltons) are indicated to the left of the gels.

FIG. 6.

Estimated copy number of VZV genomes in latently infected cotton rat ganglia from experiment 3 (Table 1). The geometric mean number of VZV genome copies per 500 ng of ganglia DNA in PCR-positive ganglia is shown at the bottom of the figure. Filled circles show the viral copy numbers for samples that exceeded the limit of detection (≥10 copies per 500 ng of DNA), while open circles represent samples whose copy numbers were below the limit of detection.

FIG. 7.

RNA transcripts corresponding to the 5′ end of ORF63 are expressed in animals latently infected with ROka63DA, ROka, and ROka63DAR. RNA was isolated from dorsal root ganglia, cDNA was prepared, and PCR was performed, followed by Southern blotting for ORF63. ORF63 transcripts were detected in animals 4 and 5 infected with ROka63DA, animals 7, 8, and 10 infected with ROka, and animals 11, 12, and 13 infected with ROka63DAR. The positions of markers showing the size of DNA (in base pairs) are indicated to the left of the gels.