Jennifer Biryukov

Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPVs capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.

Papillomavirus Infectious Pathways: A Comparison of Systems

The HPV viral lifecycle is tightly linked to the host cell differentiation, causing difficulty in growing virions in culture. A system that bypasses the need for differentiating epithelium has allowed for generation of recombinant particles, such as virus-like particles (VLPs), pseudovirions (PsV), and quasivirions (QV). Much of the research looking at the HPV life cycle, infectivity, and structure has been generated utilizing recombinant particles. While recombinant particles have proven to be invaluable, allowing for a rapid progression of the HPV field, there are some significant differences between recombinant particles and native virions and very few comparative studies using native virions to confirm results are done. This review serves to address the conflicting data in the HPV field regarding native virions and recombinant particles.

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.

Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36

The currently available nonavalent human papillomavirus (HPV) vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP) vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs) (H16.V5, H16.U4 and H16.7E) and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17–36). The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

Production and characterization of a novel HPV anti-L2 monoclonal antibody panel

The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11-200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes.

Superinfection Exclusion between Two High-Risk Human Papillomavirus Types during a Coinfection

ABSTRACT Superinfection exclusion is a common phenomenon whereby a single cell is unable to be infected by two types of the same pathogen. Superinfection exclusion has been described for various viruses, including vaccinia virus, measles virus, hepatitis C virus, influenza A virus, and human immunodeficiency virus. Additionally, the mechanism of exclusion has been observed at various steps of the viral life cycle, including attachment, entry, viral genomic replication, transcription, and exocytosis. Human papillomavirus (HPV) is the causative agent of cervical cancer. Recent epidemiological studies indicate that up to 50% women who are HPV positive (HPV+) are infected with more than one HPV type. However, no mechanism of superinfection exclusion has ever been identified for HPV. Here, we show that superinfection exclusion exists during a HPV coinfection and that it occurs on the cell surface during the attachment/entry phase of the viral life cycle. Additionally, we are able to show that the minor capsid protein L2 plays a role in this exclusion. This study shows, for the first time, that superinfection exclusion occurs during HPV coinfections and describes a potential molecular mechanism through which it occurs. IMPORTANCE Superinfection exclusion is a phenomenon whereby one cell is unable to be infected by multiple related pathogens. This phenomenon has been described for many viruses and has been shown to occur at various points in the viral life cycle. HPV is the causative agent of cervical cancer and is involved in other anogenital and oropharyngeal cancers. Recent epidemiological research has shown that up to 50% of HPV-positive individuals harbor more than one type of HPV. We investigated the interaction between two high-risk HPV types, HPV16 and HPV18, during a coinfection. We present data showing that HPV16 is able to block or exclude HPV18 on the cell surface during a coinfection. This exclusion is due in part to differences in the HPV minor capsid protein L2. This report provides, for the first time, evidence of superinfection exclusion for HPV and leads to a better understanding of the complex interactions between multiple HPV types during coinfections.

Mutations in HPV18 E1^E4 Impact Virus Capsid Assembly, Infectivity Competence, and Maturation

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.