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.Acute infection with varicella-zoster virus (VZV) causes chickenpox. The virus spreads throughout the body and infects the nervous system. Latent infection is established in dorsal root and cranial nerve ganglia, and the virus can subsequently reactivate and cause zoster (shingles). Several VZV gene products have been shown to be expressed during latency. Transcripts encoding VZV open reading frame 4 (ORF4), ORF21, ORF29, ORF62, ORF63, and ORF66 (3, 4, 10, 21) have been detected in latently infected human ganglia. ORF63 transcripts are among the most abundant VZV mRNAs expressed during latency in some studies and have been detected in 47 to 86% of human ganglia (4, 10). ORF63 mRNA is also one of the most frequently expressed viral genes in latently infected rodent ganglia (11,25).The ORF63 protein has been detected in neurons of latently infected human (10,18,20) and rodent (6, 11) ganglia. The protein is present in the cytoplasm of neurons during latency; however, during reactivation and in cell culture, the protein is present in both the nucleus and cytoplasm (18,20,25). VZV ORF63 is expressed as an immediate-early protein and is present in virions (13). While earlier studies reported conflicting results about the transregulatory activity of ORF63 (8, 14), Bontems et al. (1) found that in transient-expression assays ORF63 repressed the VZV thymidine kinase and DNA polymerase promoters. Repression of viral ...

Varicella-Zoster Virus ORF63 Inhibits Apoptosis of Primary Human Neurons

“…Although our studies are restricted to productive infection, expression of ORF63 during latent infection raises the intriguing possibility that this gene product may exert an antiapoptotic function during neuronal latency or reactivation or, as it is expressed with immediate early kinetics, during initial infection and establishment of latency within these cells. Interestingly, in a model of VZV latent infection using cotton rats, ORF63 recently has been shown to play an important role in the efficient establishment of VZV latency in these animals, although the mechanism underlying this function remains to be defined (8,9). Several novel systems for investigating the neurotropism of VZV in vivo using chimeric NOD-SCID mouse-human neural cells or intact DRG xenografts also recently have been described (4,38).…”

“…ORF63 is duplicated in the VZV genome as ORF70. Cosmid-based deletion of the diploid VZV genes ORF63/ORF70 revealed that at least one copy was necessary for VZV replication in vitro (34), although more recently, viruses with deletions in both ORF63 and ORF70 have been shown to be viable, albeit with impaired growth capabilities in cell culture (8,9). Thus, for the following studies of apoptosis in which we directly compared parental and mutant viruses, we used viruses with the single ORF63 gene deletion, rOka⌬ORF63 and rOka⌬ORF70, which have parental virus patterns of replication and plaque formation (34).…”

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

“…A recombinant VZV in which 90% of both ORF63 and its duplicate gene, ORF70, are deleted is viable but impaired for growth in cell culture and latency in cotton rats (5). Analysis of additional ORF63 mutants showed that viruses that are impaired for replication in cell culture are also impaired for latency, while mutants that are not impaired for replication establish latencies at frequencies and copy numbers similar to those of parental virus (6).…”

Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63

“…During latency, VZV expresses only a limited number of gene products. The repeated detection of open reading frame (ORF) 63 transcripts and protein in latently infected human ganglia (Cohrs et al, 1996;Mahalingam et al, 1996;Lungu et al, 1998;Cohrs et al, 2000;Kennedy et al, 2000;Grinfeld & Kennedy, 2004;Gary et al, 2006;Cohrs & Gilden, 2007), ganglia from VZV-infected guinea pigs (Chen et al, 2003) and cotton rats (Kennedy et al, 2001;Cohen et al, 2004), as well as in the simian model of VZV latency (Messaoudi et al, 2009) suggests that this gene and gene product are important for the maintenance of virus latency.…”