Christopher B. Buck

Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan

Genital human papillomavirus (HPV) infection is the most common sexually transmitted infection, and virtually all cases of cervical cancer are attributable to infection by a subset of HPVs (reviewed in ref. 1). Despite the high incidence of HPV infection and the recent development of a prophylactic vaccine that confers protection against some HPV types, many features of HPV infection are poorly understood. It remains worthwhile to consider other interventions against genital HPVs, particularly those that target infections not prevented by the current vaccine. However, productive papillomavirus infection is species- and tissue-restricted, and traditional models use animal papillomaviruses that infect the skin or oral mucosa. Here we report the development of a mouse model of cervicovaginal infection with HPV16 that recapitulates the establishment phase of papillomavirus infection. Transduction of a reporter gene by an HPV16 pseudovirus was characterized by histology and quantified by whole-organ, multispectral imaging. Disruption of the integrity of the stratified or columnar genital epithelium was required for infection, which occurred after deposition of the virus on the basement membrane underlying basal keratinocytes. A widely used vaginal spermicide, nonoxynol-9 (N-9), greatly increased susceptibility to infection. In contrast, carrageenan, a polysaccharide present in some vaginal lubricants, prevented infection even in the presence of N-9, suggesting that carrageenan might serve as an effective topical HPV microbicide.

Efficient Intracellular Assembly of Papillomaviral Vectors

ABSTRACT Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.

Merkel Cell Polyomavirus and Two Previously Unknown Polyomaviruses Are Chronically Shed from Human Skin

Mounting evidence indicates that Merkel cell polyomavirus (MCV), a circular double-stranded DNA virus, is a causal factor underlying a highly lethal form of skin cancer known as Merkel cell carcinoma. To explore the possibility that MCV and other polyomaviruses commonly inhabit healthy human skin, we developed an improved rolling circle amplification (RCA) technique to isolate circular DNA viral genomes from human skin swabs. Complete MCV genomes were recovered from 40% of healthy adult volunteers tested, providing full-length, apparently wild-type cloned MCV genomes. RCA analysis also identified two previously unknown polyomavirus species that we name human polyomavirus-6 (HPyV6) and HPyV7. Biochemical experiments show that polyomavirus DNA is shed from the skin in the form of assembled virions. A pilot serological study indicates that infection or coinfection with these three skin-tropic polyomaviruses is very common. Thus, at least three polyomavirus species are constituents of the human skin microbiome.

Carrageenan Is a Potent Inhibitor of Papillomavirus Infection

Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate–independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs.

Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays.

Merkel cell polyomavirus (MCV) is a newly‐discovered human tumor virus found in ∼80% of Merkel cell carcinoma (MCC). The rate of MCV infection among persons without MCC is unknown. We developed a MCV virus‐like particle (VLP) enzyme‐linked immunoassay (EIA) that does not cross‐react with human BK or murine polyomaviruses. Peptide mapping of the MCV VP1 gene and immunoblotting with denatured MCV VLP are less sensitive than the MCV EIA in detecting MCV antibodies suggesting antibody reactivity in this assay primarily targets conformational but not linear epitopes. Among MCC patients, all 21 (100%) patients tested with MCV‐positive tumors had high serum MCV IgG but not high MCV IgM levels. Only 3 of 6 (50%) MCC patients with MCV‐negative tumors were positive for MCV antibodies. Sera from most adults, including 107 of 166 (64%) blood donors, 63 of 100 (63%) commercial donors and 37 of 50 (74%) systemic lupus erythematosus patients, show evidence for prior MCV exposure. Age‐specific MCV prevalence was determined by examining a cross‐sectional distribution of 150 Langerhans cell histiocytosis (an unrelated neoplasm) patient sera. MCV prevalence increases from 50% among children age 15 years or younger to 80% among persons older than 50 years. We did not find evidence for vertical transmission among infants. Although past exposure to MCV is common among all adult groups, MCC patients have a markedly elevated MCV IgG response compared with control patients. Our study demonstrates that MCV is a widespread but previously unrecognized human infection.

Arrangement of L2 within the Papillomavirus Capsid

ABSTRACT Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or “split GFP” technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.

Maturation of Papillomavirus Capsids

ABSTRACT The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.

Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Most current evidence suggests that the infection of resting CD4+ T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4+ T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life = 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.

A Protective and Broadly Cross-Neutralizing Epitope of Human Papillomavirus L2

ABSTRACT We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.