Cynthia D. Thompson

The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding

Using a murine challenge model, we previously determined that human papillomavirus (HPV) pseudovirions initially bind preferentially to the cervicovaginal basement membrane (BM) at sites of trauma. We now report that the capsids undergo a conformational change while bound to the BM that results in L2 cleavage by a proprotein convertase (PC), furin, and/or PC5/6, followed by the exposure of an N-terminal cross-neutralization L2 epitope and transfer of the capsids to the epithelial cell surface. Prevention of this exposure by PC inhibition results in detachment of the pseudovirions from the BM and their eventual loss from the tissue, thereby preventing infection. Pseudovirions whose L2 had been precleaved by furin can bypass the PC inhibition of binding and infectivity. Cleavage of heparan sulfate proteoglycans (HSPG) with heparinase III prevented infection and BM binding by the precleaved pseudovirions, but did not prevent them from binding robustly to cell surfaces. These results indicate that the infectious process has evolved so that the initial steps take place on the BM, in contrast to the typical viral infection that is initiated by binding to the cell surface. The data are consistent with a dynamic model of in vivo HPV infection in which a conformational change and PC cleavage on the BM allows transfer of virions from HSPG attachment factors to an L1-specific receptor on basal keratinocytes migrating into the site of trauma.

Human α-defensins block papillomavirus infection

Sexually transmitted human papillomaviruses (HPVs) are the primary cause of cervical cancer. Recent advances in techniques for production of papillomaviral vectors [known as pseudoviruses (PsVs)] have made it possible to perform high-throughput screens for compounds that might block the initial stages of papillomavirus infection. We have used PsVs to screen a variety of compounds that might function as inhibitors of HPV infection, with emphasis on human peptides previously implicated in innate antimicrobial immunity. Little is known about the possible activity of these peptides against nonenveloped viruses, such as HPVs. Our screen revealed that human α-defensins 1-3 [known as human neutrophil peptides (HNPs) 1-3] and human α-defensin 5 (HD-5) are potent antagonists of infection by both cutaneous and mucosal papillomavirus types. In contrast, human β-defensins 1 and 2 displayed little or no anti-HPV activity. HD-5 was particularly active against sexually transmitted HPV types, with 50% inhibitory doses in the high ng/ml range. Microscopic studies of PsV inhibition by the α-defensins revealed that they block virion escape from endocytic vesicles but not virion binding or internalization. Consistent with this finding, PsVs remained susceptible to inhibition by α-defensins for many hours after initial binding to cells. HNPs 1-3 and HD-5 have been reported to be present in the female genital tract at levels that overlap those that inhibit HPVs in vitro, suggesting that they could present a natural barrier to the sexual transmission of HPV and could serve as the basis of a broad-spectrum topical microbicide.

Neutralization of Human Papillomavirus with Monoclonal Antibodies Reveals Different Mechanisms of Inhibition

ABSTRACT The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.

Production of papillomavirus-based gene transfer vectors.

Papillomaviruses are a diverse group of pathogens that infect the skin and mucosal tissues of humans and various animal species. The viral genome is a circular, double‐stranded DNA molecule ∼8‐kb in length. The non‐enveloped papillomavirus capsid is composed of a virally encoded major coat protein, L1, and a minor coat protein, L2. L1 and L2 co‐assemble when expressed in mammalian cells, and can promiscuously encapsidate essentially any <8‐kb plasmid present in the cell nucleus. In the last several years, there has been rapid development of techniques for intracellular production of papillomavirus‐based gene transfer vectors (also known as pseudoviruses). This unit outlines the production and propagative amplification of papillomaviral vectors. The system represents a highly tractable method for converting pre‐existing mammalian expression plasmids into infectious virus stocks. The resulting vectors have utility for in vitro, as well as in vivo gene delivery applications. Curr. Protoc. Cell Biol. 37:26.1.1‐26.1.19.

The Inhibition of Apoptosis in Myositis and in Normal Muscle Cells

The mechanism of injury and death of muscle cells in the inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis) remains obscure. We and others have not detected apoptosis in the muscle biopsies from patients with myositis despite clear evidence of cell damage and loss. We provide evidence in this study that Fas ligand (FasL) as well as Fas is present on muscle cells and inflammatory cells in myositis biopsies: Fas is present on most muscle cells and lymphocytes, and FasL is present on degenerating muscle cells and many infiltrating mononuclear cells. The expression of both Fas and FasL in the inflamed tissue makes the absence of apoptosis more striking. To address the mechanisms of this resistance to classical apoptosis in muscle cells, we have investigated the expression of the antiapoptotic molecule FLICE (Fas-associated death domain-like IL-1-converting enzyme)-inhibitory protein (FLIP) in muscle biopsies of myositis patients and in cultured human skeletal muscle cells. Using laser capture microscopy, we have shown that FLIP is expressed in the muscle fibers and on infiltrating lymphocytes of myositis biopsies. Furthermore, we have shown that FLIP, but not Bcl-2, is expressed in cultured human skeletal muscle cells stimulated with proinflammatory cytokines, and inhibition of FLIP with antisense oligonucleotides promotes significant cleavage of poly(ADP-ribose) polymerase autoantigen, a sensitive indicator of apoptosis. These studies strongly suggest that the resistance of muscle to Fas-mediated apoptosis is due to the expression of FLIP in muscle cells in the inflammatory environment in myositis.

Maturation of the Human Papillomavirus 16 Capsid

ABSTRACT Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. IMPORTANCE Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines. Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

NF-κB elements contribute to junB inducibility by lipopolysaccharide in the murine macrophage cell line RAW264.7

Macrophages respond to bacterial lipopolysaccharide (LPS) by activating latent cis‐acting factors that initiate transcription of immediate early genes. One such immediate early gene, junB, is induced by LPS in macrophages within 30 min. To identify elements that mediate the induction of junB by LPS, upstream and downstream sequences flanking the junB gene were examined by transient expression in the RAW264.7 murine macrophage cell line using a luciferase reporter gene vector containing the junB minimal promoter. A >10‐fold enhancement was associated with a 222 bp region downstream of the junB promoter in response to LPS. Transient reporter assays demonstrated that multiple nuclear factor (NF) κB sites are required for inducibility of junB by LPS in RAW264.7 cells. Electrophoretic mobility shift assays confirmed binding of LPS‐induced nuclear proteins included p50/p65 heterodimers at these NF‐κB sites.

Interferon Gamma Prevents Infectious Entry of Human Papillomavirus 16 via an L2-Dependent Mechanism

ABSTRACT In this study, we report that gamma interferon (IFN-γ) treatment, but not IFN-α, -β, or -λ treatment, dramatically decreased infection of human papillomavirus 16 (HPV16) pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus types, we found that many, but not all, PsV types were also inhibited by IFN-γ. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-γ, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN-γ treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN-γ treatment was mapped to the L2 protein, as determined with infectious hybrid PsV, in which the L1 protein was derived from an IFN-γ-sensitive HPV type and the L2 protein from an IFN-γ-insensitive type or vice versa. IMPORTANCE A subset of HPV are the causative agents of many human cancers, most notably cervical cancer. This work describes the inhibition of infection of multiple HPV types, including oncogenic types, by treatment with IFN-γ, an antiviral cytokine that is released from stimulated immune cells. Exposure of cells to IFN-γ has been shown to trigger the expression of proteins with broad antiviral effector functions, most of which act to prevent viral transcription or translation. Interestingly, in this study, we show that infection is blocked at the early step of virus entry into the host cell by retention of the minor capsid protein, L2, and the viral genome instead of trafficking into the nucleus. Thus, a novel antiviral mechanism for IFN-γ has been revealed.

Involvement of nucleophosmin (NPM1/B23) in assembly of infectious HPV16 capsids

We report that during assembly of HPV16 pseudovirus (PsV) the minor capsid protein, L2, interacts with the host nucleolar protein nucleophosmin (NPM1/B23). Exogenously-expressed L2 colocalized with NPM1, a complex containing both proteins, could be immunoprecipitated, and L2 could redirect to the nucleus NPM1 that was pharmacologically or genetically restricted to the cytoplasm. Coexpression of the major capsid protein, L1, prevented both the colocalization and the biochemical association, and L1 pentamers could displace L2 from L2/NPM1 complexes attached to a nuclear matrix. HPV16 PsV that was produced in a cell line with reduced NPM1 levels had significantly lower infectivity compared to PsV produced in the parental cell line, although the PsV preparations had comparable L1 and L2 ratios and levels of encapsidated DNA. The PsV produced in NPM1-deficient cells showed increased trypsin sensitivity and exhibited decreased L2 levels during endocytosis. These results suggest a critical role for NPM1 in establishing the correct interactions between L2 and L1 during HPV capsid assembly. A decrease in cellular levels of NPM1 results in the formation of seemingly normal, but unstable, capsids that result in a premature loss of L2, thus inhibiting successful infection. No role for NPM1 in HPV infectious entry was found.