Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Abstract

Papillomavirus capsids are composed of 72 pentamers reinforced through inter-and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14,15,30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14,15,30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ϳ8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a Tϭ7 icosahedral lattice (2,9,17,19,36,42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6,12,25,40,43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7,20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation...

Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[ a ]pyrene

“…HPV31b infectivity assays were performed using protocols we have previously published (2). Genome encapsidation assays were performed using protocols essentially as we have previously published (16) except that only one raft tissue per treatment was utilized for preparing crude viral preparations by Dounce homogenization. HPV31 targets were amplified using 5Ј and 3Ј primers as follows: 0. , and 10 1 serially diluted pBSHPV31 copy number controls.…”

“…In our hands, tissue growth and stratification are readily visible starting on day 8 after the dermal equivalents were lifted onto the air-liquid interface. This growth period also coincides with early steps of virion morphogenesis (16,57). When added during this time span, addition of the CDK1 inhibitor was not inhibitory to tissue growth.…”

“…This assay was based on the expectation that all viruses produced in untreated tissues are infectious and are units of encapsidated viral genomes. HPV31b raft tissues were treated with 1 M BaP and/or cotreated with 10 nM purvalanol A as described above, harvested, and used for preparing viral stocks using protocols we have previously described (16). Viral stocks were incubated with benzonase for digestion of exogenous cellular DNA and viral DNA, and viral genomes were extracted from capsids also as previously described (16).…”

“…To examine the possibility that the failure of virus that had been treated with BaP and purvalanol A to infect HaCaT cells was due to either disassembly or lack of assembly of these particles in response to inhibition of CDK1 kinase activity, a qPCR-based DNA encapsidation assay was used to quantify total endonuclease-resistant viral genomes using methods we have previously described (16). This assay was based on the expectation that all viruses produced in untreated tissues are infectious and are units of encapsidated viral genomes.…”

“…HPV31b raft tissues were treated with 1 M BaP and/or cotreated with 10 nM purvalanol A as described above, harvested, and used for preparing viral stocks using protocols we have previously described (16). Viral stocks were incubated with benzonase for digestion of exogenous cellular DNA and viral DNA, and viral genomes were extracted from capsids also as previously described (16). We have previously reported that 2) were used for preparing crude viral stocks followed by infection of HaCaT cells as we have described previously (2).…”