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The genomes of human and animal papillomaviruses amplify in keratinocytes undergoing terminal squamous differentiation. Two approaches have been developed to facilitate the investigation into the requirement for viral DNA replication and its regulation outside the context of the host tissues. Under these conditions, the investigation can be conducted independently of viral genes required to re-establish the S-phase milieu in the differentiated keratinocytes to support viral DNA replication. The first method is transient replication in cell lines after transfection of a plasmid containing the viral origin of replication together with expression vectors of the necessary viral proteins that direct specific initiation from the origin. The second is cell-free replication in which purified viral replication proteins are complemented by cell extracts to initiate origin-specific replication. The two methods have identified the origin, the viral E1 and E2 proteins necessary for initiation, their functions, and the host factors that are required to support and regulate the viral DNA replication. These two methods are complementary in providing answers and insights that either one alone may not be able to achieve. This chapter provides a practical guide to these two replication assays. [ABSTRACT FROM AUTHOR]

The productive phase of human papillomavirus (HPV) infection is dependent on squamous differentiation of epithelial keratinocytes. Organotypic culture systems of primary human keratinocytes (PHKs) or immortalized keratinocytes that contain HPV genomes were developed to recapitulate this permissive environment. A complementary approach to determine the functions of individual HPV genes and to examine the virus-host interactions is to introduce the gene, alone or in combination, into keratinocytes that are then grown in organotypic cultures. The success of the latter approach depends on the methodology of retrovirus-mediated gene transfer, which can transduce the viral gene or genes into an entire population of PHKs. In this chapter, we describe the strategies and methods of retrovirus-mediated gene transfer into keratinocytes grown into organotypic cultures. [ABSTRACT FROM AUTHOR]

The use of tyramide-coupled immunofluorescence at the single cell level provides expedient, clean, and sensitive signals for detection of DNA, RNA, or proteins. The principle is based on the ability of horseradish peroxidase (HRP) to cleave tyramides into a free radical species with a very short diffusion radius. The free radicals are then covalently bound to electron-rich moieties such as tyrosine in proteins proximal to the targets. Here we present protocols for tyramide fluorescent in situ hybridization (T-FISH), which detects unique DNA species using DNA probes as short as approx 300-500 bp, or unique RNA species with probes as small as an oligonucleotide. We also present a protocol for tyramide immunofluorescence (T-IF) to detect protein antigens. By combining these protocols with several tyramide-coupled fluorophores, multiple targets can be detected simultaneously in situ, which is ideal for in-depth analyses at the molecular and cellular levels. Finally, we describe the detection of nascent viral RNA transcripts simultaneously with integrated viral genomes or chromosomal domains in single cells or tissue sections. [ABSTRACT FROM AUTHOR]

Messenger RNAs of higher eucaryotes are usually modified posttran-scriptionally to contain 5′ caps and nucleotide methylation, 3′ polyadenylation, and one or more internal splices to remove introns and join exon segments into the mature protein-coding sequences. With the abilities of retroviral reverse transcriptases to create complementary DNA (cDNA) copies of RNA and of thermostable DNA polymerases to amplify specific DNA segments by repeated cycles of denaturation of duplex templates, annealing of complementary oligonucleotide primers, and strand elongation, it is becoming increasingly popular to use this highly sensitive polymerase chain reaction (PCR) to isolate partial cDNA sequences for the purpose of identifying RNA splice sites and inferring the coding capacity. The splice junction information from the partial cDNAs, together with additional biophysical or biochemical information, can then be employed to map the 5′ ends of the mRNAs and assign the AUG protein initiation codon and open reading frame to the message. [ABSTRACT FROM AUTHOR]