Absence or Overexpression of the Varicella-Zoster Virus (VZV) ORF29 Latency-Associated Protein Impairs Late Gene Expression and Reduces VZV Latency in a Rodent Model

Abstract

Varicella-zoster virus (VZV) ORF29 encodes the viral single-stranded DNA binding protein and is expressed during latency in human ganglia. We constructed an ORF29 deletion mutant virus and showed that the virus could replicate only in cells expressing ORF29. An ORF29-repaired virus, in which ORF29 was driven by a cytomegalovirus promoter, grew to peak titers similar to those seen with the parental virus. The level of ORF29 protein in cells infected with the repaired virus was greater than that seen with parental virus. Infection of cells with either the ORF29 deletion or repaired virus resulted in similar levels of VZV immediate-early proteins but reduced levels of glycoprotein E compared to those observed with parental virus. Cotton rats infected with the ORF29 deletion mutant had a markedly reduced frequency of latent infection in dorsal root ganglia compared with those infected with parental virus (P < 0.00001). In contrast, infection of animals with the ORF29 deletion mutant resulted in a frequency of ganglionic infection at 3 days similar to that seen with the parental virus. Animals infected with the ORF29-repaired virus, which overexpresses ORF29, also had a reduced frequency of latent infection compared with those infected with parental virus (P ‫؍‬ 0.0044). These studies indicate that regulation of ORF29 at appropriate levels is critical for VZV latency in a rodent model. ORF29 encodes a 130-kDa protein that binds to singlestranded DNA and localizes predominantly to the nucleus of virus-infected cells in vitro (24). ORF29 contains an atypical nuclear localization signal within amino acids 9 to 154, and transport to the nucleus requires Ran and karyopherins (37). While ORF29 protein is present in the nucleus of lytically infected cells, the protein is in the cytoplasm of neurons from human ganglia (17,25). ORF29 protein localizes to the cytoplasm of guinea pig enteric ganglia neurons (5) and in an astrocyte-like cell line (38). Treatment with a proteosome inhibitor or expression of herpes simplex virus type 1 (HSV-1) ICP0 or VZV ORF61 results in translocation of ORF29 protein to the nucleus in both guinea pig enteric ganglia neurons and the astrocyte-like cell line. Primary infection with varicella-zoster virus (VZV) results inORF29 protein is secreted from VZV-infected fibroblasts and is endocytosed by human neurons in vitro (1). The protein is present in endothelial and epithelial cells in the skin of patients with varicella and zoster; the protein is also located in nerves in the dermis of patients with varicella. ORF29 protein is not present in virions (23).ORF29 is expressed from a bidirectional promoter that it shares with ORF28 (27,28,40). Expression is activated during lytic infection by ORF62 protein and the USF transcription factor. During lytic infection, ORF29 transcripts of 4.1 and 4.2 kb are expressed, while during latency only the 4.2-kb transcript is present (26). Expression of ORF29 in T cells enhances the ability of ORF62 protein to transactivate the gI promoter; however, the effect of ...

A Sequence within the Varicella-Zoster Virus (VZV) OriS Is a Negative Regulator of DNA Replication and Is Bound by a Protein Complex Containing the VZV ORF29 Protein

“…This suggestion follows from the findings of Cohen et al (11), who found that the overexpression of the ORF29 protein impaired the establishment of latency in a rodent model. Some limited replication of VZV is believed to occur in DRG neurons prior to the establishment of latency, resulting in the presence of multiple copies of the viral genome per neuron (27).…”

“…The ORF29 protein is believed to be essential for the initiation of viral DNA replication by analogy with ICP8 (20,30). ORF29 transcripts and protein have been reported to be present in latently infected human dorsal root ganglion (DRG) neurons (36), and ORF29 is involved in the establishment of latency in the cotton rat model (11).…”

Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

“…VZV ORF 18 encodes the small subunit of ribonucleotide reductase, an enzyme critical to synthesis of DNA precursors, and also protects neurons from apoptotic cell death (34). VZV ORF 21 encodes a nucleocapsid protein (29), and VZV ORF 29 encodes a DNA binding protein that is essential for virus replication (4,31). VZV ORFs 40 and 68 encode virus structural proteins.…”

Infected peripheral blood mononuclear cells transmit latent varicella zoster virus infection to the guinea pig enteric nervous system

“…DNA encoding all 4 of the ORFs examined was detected at 28 days in both the colon and ileum in each of 4 guinea pigs; nevertheless, transcripts encoding ORFs 40 and 67 could not be detected in 3 of the 4 animals. Transcripts encoding ORFs 29 and 66, however, which encode genes transcribed during latency (Chen et al , 2011; Cohen et al , 2007; Cohrs et al , 2003; Folster et al , 2011; Grinfeld and Kennedy, 2004; Sato et al , 2003b; Stallings et al , 2006), were found in the colon and ileum of all 4 guinea pigs. These observations are consistent with the idea that VZV established enteric latency in 3/4 guinea pigs and that an asymptomatic lytic infection, associated with the expression of ORFs 40 and 67, was present in the bowel of 1 of the 4 animals.…”