First FASEB conference in Africa-The 2023 FASEB conference on Imaging Cellular and Chromosome Dynamics held on August 27-30, 2023 in Accra, Ghana.

Access to scientific meetings and conferences is limited in low- and middle-income countries (LMIC). Efforts are being implemented to rectify this issue through short workshops, seminars, and conferences. Sena Institute of Technology (SIT), a nonprofit research institute based in Ghana, is one such organization championing this initiative. Through a partnership with the Federation of American Societies for Experimental Biology (FASEB), SIT hosted the first FASEB conference in Africa from August 27-30, 2023 in Ghana. The 3-day conference brought together scientists specialized in imaging, genetics, and cell biology from across the globe to discuss the theme "Imaging Cellular and Chromosome Dynamics." The Ghanaian Ministry of Environment, Science, Technology and Innovation (MESTI) and the Ghana Tourism Authority (GTA) provided local support to the meeting. At the end of the conference, participants recommended continuing engagement and the organization of more such meetings on the African continent.

Vorinostat, a pan-HDAC inhibitor, abrogates productive HPV-18 DNA amplification.

Human papillomaviruses (HPVs) cause epithelial proliferative diseases. Persistent infection of the mucosal epithelia by the high-risk genotypes can progress to high-grade dysplasia and cancers. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and histone deacetylases (HDACs) that remodel chromatin and regulate gene expression. HDACs are also essential to remodel and repair replicating chromatin to enable the progression of replication forks. As such, Vorinostat (suberoylanilide hydroximic acid), and other pan-HDAC inhibitors, are used to treat lymphomas. Here, we investigated the effects of Vorinostat on productive infection of the high-risk HPV-18 in organotypic cultures of primary human keratinocytes. HPV DNA amplifies in the postmitotic, differentiated cells of squamous epithelia, in which the viral oncoproteins E7 and E6 establish a permissive milieu by destabilizing major tumor suppressors, the pRB family proteins and p53, respectively. We showed that Vorinostat significantly reduced these E6 and E7 activities, abrogated viral DNA amplification, and inhibited host DNA replication. The E7-induced DNA damage response, which is critical for both events, was also compromised. Consequently, Vorinostat exposure led to DNA damage and triggered apoptosis in HPV-infected, differentiated cells, whereas uninfected tissues were spared. Apoptosis was attributed to highly elevated proapoptotic Bim isoforms that are known to be repressed by EZH2 in a repressor complex containing HDACs. Two other HDAC inhibitors, Belinostat and Panobinostat, also inhibited viral DNA amplification and cause apoptosis. We suggest that HDAC inhibitors are promising therapeutic agents to treat benign HPV infections, abrogate progeny virus production, and hence interrupt transmission.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections.

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90-99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.

Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid- and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures.

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.

Human papillomavirus (HPV) E7 induces prolonged G2 following S phase reentry in differentiated human keratinocytes.

The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G(2) phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181-194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862-4873), also elicited extensive G(2) responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G(2)/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr(14) or Tyr(15), and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser(216), unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser(1981)) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G(2) phase in the differentiated cells following S phase induction.

Nonconserved lysine residues attenuate the biological function of the low-risk human papillomavirus E7 protein.

The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures.

PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro.

BACKGROUND: High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation. METHODS: Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting. RESULTS: Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10. CONCLUSION: The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.

Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6.

MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancer-derived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR-34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16(+) and HPV18(+) cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G(0)/G(1) phase cells. Ectopic expression of miR-34a in HPV18(+) HeLa cells and HPV(-) HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer.

Anal-rectal cytology: correlation with human papillomavirus status and biopsy diagnoses in a population of HIV-positive patients.

OBJECTIVES: We describe the cytological distribution of disease, correlate cytological diagnoses with human papillomavirus (HPV) DNA status and surgical biopsy diagnoses, determine if CD4 counts correlate with lesion severity, and compare anal-rectal data of HIV-infected patients (primarily men) with cervical data. MATERIALS AND METHODS: A retrospective search of the computerized database identified 118 HIV-positive patients who had anal-rectal cytology. Cytology results were compared with available follow-up data including repeat anal-rectal cytology tests, surgical biopsy, CD4 counts, and HPV DNA polymerase chain reaction-based genotyping. RESULTS: Cytological diagnoses included 3% unsatisfactory for diagnosis, 41% negative for intraepithelial lesion or malignancy (NILM), 23% atypical squamous cells of undermined significance (ASC-US), 31% low-grade squamous intraepithelial lesion (LSIL), and 2% high-grade squamous intraepithelial lesion (HSIL) (ASC-US/squamous intraepithelial lesion, 0.7:1). Two anal intraepithelial neoplasia (AIN) II, 10 AIN III, and 1 invasive squamous cell carcinoma were histologically detected (11%). The majority of AIN II was preceded by LSIL, 54%; ASC-US, 15%; and HSIL, 8%. The false-negative fraction was 23%. Sensitivity, specificity, negative predictive value, and positive predictive value were 92%, 8%, 33%, and 67%, respectively. Of those HPV tested concurrent with the first cytology specimen, 48% NILM, 78% ASC-US, and 100% LSIL were HPV positive. Mean CD4 counts (per microliter) were lower in patients with HSIL (243 [SD, 65]) compared with LSIL (400 [SD, 261]) and NILM (428 [SD, 232]). CONCLUSIONS: Anal-rectal cytology is a useful screening test. A high percentage of AIN II lesions were detected in this at-risk population, and the majority was detected following cytological abnormality.

Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes.

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification.

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds.

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-beta(1) and TGF-beta(2) precursor proteins, but an increase in expression of TGF-beta(3) precursor protein and TGF-beta type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein.

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.

Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes.

The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2-4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12-16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

Genomic diversity and phylogenetic relationships of human papillomavirus 16 (HPV16) in Nepal.

OBJECTIVES/BACKGROUND: Sequence variants in HPV16 confer differences in oncogenic potential; however, to date there have not been any HPV sequence studies performed in Nepal. The objective of this study was to characterize HPV16 viral genome sequences from Nepal compared to a reference sequence in order to determine their lineages. Additionally, we sought to determine if five High-grade Squamous Intraepithelial Lesion (HSIL) subjects were genetically distinct from the non-HSIL subjects. METHODS: DNA was isolated from exfoliated cervical cells from 17 individuals in Nepal who were previously identified to be HPV16-positive. A custom HPV16 Ion Ampliseq panel of multiplexed degenerate primers was designed that generated 47 overlapping amplicons and covered 99% of the viral genome for all known HPV16 variant lineages. All sequence data were processed through a custom quality control and analysis pipeline of sequence comparisons and phylogenetic analysis. RESULTS: There were high similarities across the genomes, with two major indels observed in the non-coding region between E5 and L2. Compared to the PAVE reference HPV16 genome, there were up to 9, 4, 38, 27, 8, 7, 52, and 32 nucleotide variants in the E6, E7, E1, E2, E4, E5, L2, and L1 genes in the Nepalese samples, respectively. Based on sequence variation, HPV16 from Nepal falls across the A, C, and D lineages in this study. We found no evidence of genetic distinctness between HSIL and non-HSIL subjects. CONCLUSIONS: The evolutionary and pathological characteristics of the representative HPV16 genomes from Nepal seem similar to results from other parts of the world and provide the basis for further studies.

Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9-[(2-Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence.

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.

Retrovirus-mediated gene transfer to analyze HPV gene regulation and protein functions in organotypic "raft" cultures.

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.