Mutational Analysis of Open Reading Frames 62 and 71, Encoding the Varicella-Zoster Virus Immediate-Early Transactivating Protein, IE62, and Effects on Replication In Vitro and in Skin Xenografts in the SCID-hu Mouse In Vivo

Abstract

The varicella-zoster virus (VZV) genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in U S allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant Oka⌬62 (rOka⌬62) or rOka⌬71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.Varicella-zoster virus (VZV) belongs to the alphaherpesvirus subfamily of the Herpesviridae. VZV is the causative agent of varicella, which is characterized by cell-associated viremia and a cutaneous vesicular rash (4). VZV establishes latency in cells within sensory ganglia during primary infection. VZV reactivation from latency results in herpes zoster, a localized skin rash in the distribution of nerves from the affected ganglion. VZV is the first human herpesvirus for which a vaccine has been developed to prevent primary infection (58). This live attenuated varicella vaccine was created by serial passage of a wild-type clinical isolate, the parent Oka (pOka) strain, in guinea pig embryo cells and human fibroblasts to generate the varicella vaccine virus (vOka).The VZV genome consists of approximately 125 kb and has at least 70 unique open reading frames (ORFs) (52). As is characteristic of herpesviruses, the double-stranded DNA genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three duplicated genes, ORF62/71, ORF63/ 70, and ORF64/69, are located in repeats at each end of the U S segment. The two VZV origins of replication, designated OriS, are also located in these repeat regions. The immediate-early 62 (IE62) protein,...

Varicella-zoster virus infection of human neural cells in vivo

“…VZV gene expression in neural cells differed dramatically from VZV infection of epidermal and dermal cells in human skin in vivo, which is associated with cell destruction and release of infectious virions (20)(21)(22)(23)(24). IE62, the major viral transactivating protein and an important component of the virion tegument (38,39), was restricted to the nuclei of neurons and glial cells, whereas IE62 localized to the cytoplasm of infected skin cells. IE63 was abundant in the cytoplasm of neurons and glial cells, whether expressed by low-passage pOka or the single-copy rOka⌬70 recombinant, which differed from its predominant nuclear expression in VZV-infected skin cells.…”

“…VZV causes lytic infection of human dermal and epidermal cells in skin xenografts in vivo (20)(21)(22)(23)(24)38). To compare VZV protein expression in human neural and skin cells in vivo, skin xenografts were collected 3 weeks after VZV infection, and alternate sections were examined for IE62, IE63, ORF47 kinase, or gE synthesis (Fig.…”

The Immediate-Early 63 Protein of Varicella-Zoster Virus: Analysis of Functional Domains Required for Replication In Vitro and for T-Cell and Skin Tropism in the SCIDhu Model In Vivo

“…10B). Infectious rOKA/ ORF63rev[S185] was not recovered at 20 days, but this pattern was due to extensive infection and depletion of T cells, by immunohistochemistry analysis (data not shown); declining titers at days 21 to 28 days after T-cell infection with low passage clinical isolates or intact recombinants made from cosmids is characteristic of VZV replication in T-cell xenografts (3,13,32,40).…”

“…8A) (Fig. 8B) after infection; viral protein loading was adjusted based on detection of equivalent amounts of IE4 (40). The viral protein expression profiles of the IE63 mutants were compared to rOKA, which has two intact copies of ORF63, and to rOKA/ORF63rev, which has one intact copy of ORF63 (Fig.…”

Postherpetic Neuralgia: From Preclinical Models to the Clinic

“…44 Viral infection in the sensory dorsal root ganglia (DRG) is confirmed by the presence of the immediate early protein VZV IE62, a protein that is expressed during latency and that is known to be essential for VZV replication and expression in host cells. 41,45,46 This protein has been shown to be associated with subpopulations of DRG neurons. 41 Inoculation with different viral strains.…”