The Cellular Localization Pattern of Varicella-Zoster Virus ORF29p Is Influenced by Proteasome-Mediated Degradation

Abstract

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29pis localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.Chicken pox (varicella) is a manifestation of lytic infection of cutaneous epithelial cells by varicella-zoster virus (VZV), a ubiquitous human alphaherpesvirus. Following lytic infection, VZV establishes latency in the sensory ganglia and can reactivate later during the host's life to cause shingles (zoster), a painful and potentially debilitating disease that may lead to postherpetic neuralgia (1). Recrudescence occurs when cellmediated immunity is decreased, as seen in the elderly and immunocompromised individuals, where it is associated with significant morbidity that may not be relieved by antiviral and analgesic therapy (63). An understanding of the cellular and viral factors that govern VZV latency and reactivation is critical to developing preventive methods and potential therapies for zoster and postherpetic neuralgia.During latency, viral DNA replication, late protein expression, and virion assembly do not occur. It has been reported that transcripts corresponding to the immediate-early and early open reading frames (ORFs) ORF4, -21, -29, -62, -63, and -66 are present in neurons harboring latent VZV (9-13, 15, 25, 36, 50). Proteins expressed by these ORFs are present in the cytoplasm of latently infected neurons (9,12,15,36,49,50). In particular, Lungu et al. demonstrated ...

Nuclear Import of the Varicella-Zoster Virus Latency-Associated Protein ORF63 in Primary Neurons Requires Expression of the Lytic Protein ORF61 and Occurs in a Proteasome-Dependent Manner

“…The findings in this report coupled with previously published data (59) show that localization of VZV LAPs is regulated in EN by expression of the VZV lytic protein ORF61p. Stallings et al demonstrated that ORF61p expression caused nuclear accumulation of autonomously expressed ORF29p in EN and U373MG cells.…”

“…Stallings et al previously demonstrated that latent VZV infection of EN can be reactivated after infection with an adenovirus expressing ORF61p. Upon the reactivation of VZV, LAPs such as ORF29p and ORF62p accumulate in the nucleus (59). Accordingly, we tested whether the expression of ORF61p was sufficient to cause the nuclear import of ORF63p in EN.…”

“…VZV infection of primary guinea pig enteric neuronal cultures provides a potential model for studying latency (8,59). Following infection of enteric neurons (EN), VZV expresses a subset of LAPs, and these proteins localize exclusively in the cytoplasm.…”

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

“…ORF29 protein is present in the nucleus of lytically infected cells but in the cytoplasm of human neurons during latency (17,25). Interestingly, when an astrocytoma-derived cell line is infected with adenovirus which expresses ORF29, the protein is expressed in the cytoplasm; however, when these cells are treated with a proteosome inhibitor, the half-life of ORF29 protein is increased and the protein migrates to the nucleus (38). Thus, it is possible that overexpression of ORF29 protein in ROka29DR-infected neurons could result in both cytoplasmic and nuclear expression of the protein in the cells and thereby impair latency.…”

“…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.…”