Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus.Papillomaviruses are small icosahedral viruses with doublestranded, circular DNA genomes that infect the stratified squamous epithelial tissues of vertebrates. The infection commonly results in a papilloma, or wart, formation. A small subset of the human-specific genotypes termed the high-risk human papillomaviruses (HPVs), including HPV31, have the capacity to cause cancer, most notably cervical cancer (35). Understanding the papillomaviral life cycle may allow us to establish the means to inhibit high-risk papillomaviral infections and thereby prevent HPV-associated cancers. In this study, we investigated the role the minor capsid protein, L2, plays in the papillomaviral life cycle.The life cycle of papillomaviruses is intricately tied to the differentiation of their host tissue, stratified squamous epithelium (23). In the poorly differentiated basal cells, which comprise the proliferating compartment of the epithelium and where papillomaviral infection is thought to arise, the viral genome takes up residence as a stable, nuclear plasmid that is maintained at low copy number. Only a subset of viral genes, the early genes, is selectively expressed in the basal compartment, and therefore no new virus is made there. Consequently, we refer to the infective state in the basal compartment as the nonproductive phase of the life cycle. As basal cells proliferate and undergo cell division, daughter cells that lose contact with the underlying basement membr...

Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low-and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by ␣6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.

Purpose To investigate abandonment rate of prescribed low vision devices for near tasks and factors associated with abandonment in a U.S. outpatient population. Methods A telephone survey was administered to 88 patients with low vision from four clinical sites approximately one year after examination and prescription of devices. Patients were surveyed on timing and frequency of use and reasons for abandonment of devices. The main outcome measure (abandonment) was defined as patient report of no use of prescribed device in the previous three months. Multivariate logistic regression was used to investigate significant vision and demographic factors related to abandonment. Results Of 119 prescribed devices, 19% (95% CI, 12–26) had not been used within the previous three months. Mean (±SD) better eye visual acuity at examination was 0.61±0.29 logMAR and mean age was 77±17 years. Mean time between device prescription and survey was 11±3 months. Device abandonment was not associated with age (p = 0.863), time since prescription (p = 0.125), visual acuity (p = 0.804), or category of magnification device (spectacle, handheld, stand, or video) (p = 0.412). There was a significant association between documented non-central visual field loss and abandonment of magnification device (p=0.046). Repeat administration of the survey resulted in the same abandonment classification in 15 of 15 patients (100%). Conclusions Abandonment rate was similar for this outpatient population to those previously reported in the U.S. veteran inpatient population and in other countries. Patients with visual field loss may be more likely to abandon prescribed devices.

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Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitrowherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.