Eric J. Ryndock

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.

A risk for non-sexual transmission of human papillomavirus?

Human papillomaviruses (HPVs) are estimated to be the most common sexually transmitted virus in humans. The virus is of great interest as it is the etiological agent of cervical cancer. Sexual transmission of HPV is generally accepted, however, non-sexual transmission of the virus is often debated. Here, we review the evidence from basic research and clinical studies that show HPV can survive well outside of its host to potentially be transmitted by non-sexual means. In doing so, we hope to discover problems in current prevention practices and show a need for better disinfectants to combat the spread of HPV.

Susceptibility of HPV16 and 18 to High Level Disinfectants Indicated for Semi-Critical Ultrasound Probes

Ultrasound probes used in endocavitary procedures have been shown to be contaminated with high‐risk HPV after routine use and HPV is also known to be resistant to some high level disinfectants (HLDs). This study compared efficacy of two leading ultrasound probe HLD methods; liquid ortho‐phthalaldehyde (Cidex® OPA) and an automated device using sonicated hydrogen peroxide (trophon® EPR) against HPV16 and HPV18 in a hard‐surface carrier test. Native HPV16 and HPV18 virions were generated in organotypic epithelial raft cultures. Viral lysates were dried onto carriers with a 5% (v/v) protein soil. Efficacy tests were performed against the automated device at 35% and 31.5% H2O2 and 0.55% OPA in quadruplicate with matched input, neutralization, and cytotoxicity controls. Hypochlorite was included as a positive control. Infectivity was determined by the abundance (qRT‐PCR) of the spliced E1^E4 transcript in infected recipient cells. The automated HLD device showed excellent efficacy against HPV16 and HPV18 (>5 log10 reductions in infectivity) whereas OPA showed minimal efficacy (<0.6 log10 reductions). While HPV is highly resistant to OPA, sonicated hydrogen peroxide offers an effective disinfection solution for ultrasound probes. Disinfection methods that are effective against HPV should be adopted where possible. J. Med. Virol. 88:1076–1080, 2016.

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

ABSTRACT Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16INK4 and p27KIP1, which normally mediate pRb hypophosphorylation. In contrast, p21WAF1 and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.

Roles for Human Papillomavirus Type 16 L1 Cysteine Residues 161, 229, and 379 in Genome Encapsidation and Capsid Stability

Human papillomavirus (HPV) capsids are formed through a network of inter- and intra-pentameric hydrophobic interactions and disulfide bonds. 72 pentamers of the major capsid protein, L1, and an unknown amount of the minor capsid protein, L2, form the structure of the capsid. There are 12 conserved L1 cysteine residues in HPV16. While C175, C185, and C428 have been implicated in the formation of a critical inter-pentameric disulfide bond, no structural or functional roles have been firmly attributed to any of the other conserved cysteine residues. Here, we show that substitution of cysteine residues C161, C229, and C379 for serine hinders the accumulation of endonuclease-resistant genomes as virions mature within stratifying and differentiating human epithelial tissue. C229S mutant virions form, but are non-infectious. These studies add detail to the differentiation-dependent assembly and maturation that occur during the HPV16 life cycle in human tissue.

Native human papillomavirus production, quantification, and infectivity analysis.

In a natural infection, human papillomavirus (HPV) replicates in a stratified and differentiated epithelium. We have developed an in vitro organotypic raft culture system that allows researchers to study HPV in its natural environment. Not only does this system reproduce the differentiation-dependent replication cycle of HPV, but it also allows for the production of high titers of native HPV virions. Currently, much of the HPV research has been done utilizing synthetic particles produced in transfection systems. However, by production of native virions, this research can now be continued using native particles. This chapter presents methods for producing, titering, and qualitating, via infectivity assay, native virus produced from organotypic raft culture.

Comparison of human papillomavirus type 16 replication in tonsil and foreskin epithelia.

Human papillomavirus (HPV) is well recognized as a causative agent for anogenital and oropharyngeal cancers, however, the biology of HPV infection at different mucosal locations, specifically the oral cavity, is not well understood. Importantly, it has yet to be determined if oral tissues are permissive for HPV infection and replication. We investigated for the first time the titers, infectivity, and maturation of HPV16 in oral epithelial versus genital epithelial tissue. We show that infectious HPV16 virions can be produced in oral tissue. This demonstrates, for the first time, that infectious virus could be spread via the oral cavity. HPV16 derived from oral tissue utilize a tissue-spanning redox gradient that facilitates the maturation of virions over time. Maturation is manifested by virion stability and increased susceptibility to neutralization with anti-HPV16 L1 antibodies. However, susceptibility to neutralization by anti-HPV16 L2 specific antibodies decreases during the maturation of HPV16 virions in oral tissue.

Replication of Human Papillomavirus in Culture

Human papillomaviruses (HPV) are the major factor in causing cervical cancer as well as being implicated in causing oral and anal cancers. The life cycle of HPV is tied to the epithelial differentiation system, as only native virus can be produced in stratified human skin. Initially, HPV research was only possible utilizing recombinant systems in monolayer culture. With new cell culture technology, systems using differentiated skin have allowed HPV to be studied in its native environment. Here, we describe current research studying native virions in differentiated skin including viral assembly, maturation, capsid protein interactions, and L2 cross-neutralizing epitopes. In doing so, we hope to show how differentiating skin systems have increased our knowledge of HPV biology and identify gaps in our knowledge about this important virus.