Mutational Analysis of the Repeated Open Reading Frames, ORFs 63 and 70 and ORFs 64 and 69, of Varicella-Zoster Virus

Abstract

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.Varicella-zoster virus (VZV) is a ubiquitous human herpesvirus that causes varicella during primary infection of susceptible individuals (2). VZV is a lymphotropic virus, with the capacity to infect CD4 and CD8 T cells, permitting its spread to mucocutaneous sites and producing the vesicular rash commonly referred to as chicken pox. VZV is a member of the alphaherpesvirus group and also exhibits the neurotropism characteristic of these viruses; it establishes latency in sensory nerve ganglia, and its reactivation results in herpes zoster, a localized dermatomal exanthem.The VZV genome is a double-stranded DNA molecule with open reading frames (ORFs) that are known or predicted to encode at least 69 distinct gene products. The genome consists of two main coding regions, the unique long (U L ) and unique short (U S ) segments, each of which is flanked by internal repeat (IR) and terminal repeat (TR) sequences. Functions have been assigned to only about half of the VZV gene products, and many of these are presumed because of their partial sequence homologies with herpes simplex virus type 1 (HSV-1), which is the prototype of the alphaherpesviruses. Whereas generating mutant ...

Varicella-zoster virus infection of human neural cells in vivo

“…These experiments demonstrated that VZV spreads efficiently between neural cells in vivo, without changing cellular morphology or expression of neural cell proteins, including hNCAM, hGFAP, and ␤-tubulin III. 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.…”

“…NOD-SCID mice transplanted with human neurosphere cells (StemCells Inc.) were inoculated with VZV-infected human melanoma cells at 4-6 months after transplant. Viruses tested were pOka, live attenuated vOka (Merck), and rOka⌬70, a deletion mutant with only one copy of the gene encoding IE63 (24). Melanoma cells were grown in tissue culture medium (DMEM, GIBCO) supplemented with heat-inactivated FCS.…”

“…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 expanding populations migrate throughout the brain from the SVZ to the olfactory bulb, express human neuronal and glial cell markers, and develop axonal processes. Skin and thymus͞liver xenografts in SCID mice permit analysis of VZV tropism for human epidermal and dermal cells and T cells within their intact tissue microenvironments in vivo (20)(21)(22)(23)(24). Therefore, we used the chimeric NOD-SCID mouse-human neural cell model to investigate VZV infection of differentiated neural cells in vivo.…”

“…We also evaluated a VZV deletion mutant, rOka⌬70, that lacks one copy of the gene encoding IE63, because neural cell expression of IE63 has been prominent in autopsy and animal studies (9,13,15). This mutant, and two other single-copy ORF63͞70 variants, exhibits no deficiencies in cell culture and is as virulent as the parent virus in human skin xenografts in SCID mice (24), These experiments in the NOD-SCID mouse-human neural cell model showed that VZV infection with pOka, vOka, and rOka⌬70 was characterized by efficient spread in human neurons and glial cells, without disrupting cell morphology or expression of neural cell adhesion molecules (hNCAMs), glial fibrillary acidic protein (hGFAP), or neuron-specific class III ␤-tubulin in vivo. VZV infection of human neural cells exhibited a distinctive intracellular distribution of the IE regulatory proteins and a block in synthesis of the major envelope glycoprotein, gE, compared to lytic VZV infection of epidermal and dermal cells in vivo.…”