Cyclic AMP-Dependent Protein Kinase Exhibits Antagonistic Effects on the Replication Efficiency of Different Human Papillomavirus Types.

Several types of widespread human papillomaviruses (HPVs) may induce the transformation of infected cells, provoking the development of neoplasms. Two main genera of HPVs are classified as mucosatropic alphapapillomaviruses and cutaneotropic betapapillomaviruses (α- and ß-HPVs, respectively), and they both include high-risk cancer-associated species. The absence of antiviral drugs has driven investigations into the details of the molecular mechanisms of the HPV life cycle. HPV replication depends on the viral helicase E1 and the transcription factor E2. Their biological activities are controlled by numerous cellular proteins, including protein kinases. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of α-HPV11, α-HPV18, and ß-HPV5. PKA stimulates the replication of both α-HPVs studied but has a more profound effect on the replication of high-risk α-HPV18. However, the replication of ß-HPV5 is inhibited by activated PKA in human primary keratinocytes and U2OS cells. We show that the activation of PKA signaling by different pharmacological agents induces the rapid proteasomal degradation of the HPV5 E2 protein, which in turn leads to the downregulation of E2-dependent transcription. In contrast, PKA-stimulated induction of HPV18 replication is the result of the downregulation of the E8^E2 transcript encoding a potent viral transcriptional inhibitor together with the rapid upregulation of E1 and E2 protein levels. IMPORTANCE Several types of human papillomaviruses (HPVs) are causative agents of various types of epithelial cancers. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of various types of HPVs during the initial amplification and maintenance phases of the viral life cycle. The replication of the skin cancer-related pathogen HPV5 is suppressed, whereas the replication of the cervical cancer-associated pathogen HPV18 is activated, in response to elevated PKA activity. To inhibit HPV5 replication, PKA targets the viral transcriptional activator E2, inducing its rapid proteasomal degradation. PKA-dependent stimulation of HPV18 replication relies on the downregulation of another E2 gene product, E8^E2, which encodes a potent transcriptional repressor. Our findings highlight, for the first time, protein kinase-related mechanistic differences in the regulation of the replication of mucosal and cutaneous HPV types.

Molecular detection of oncogenic subtypes of human papillomavirus (HPV) in a group of women in the Amazon region of Brazil

The natural history of cervical cancer is strongly related to the presence of human papillomavirus (HPV) infection, with its relationship with cervical cancer being a matter of concern. It is estimated that 70% of all cervical cancers worldwide are caused by HPV 16 and 18. Accordingly, the present study aimed to contribute to the identification of HPV subtypes circulating in a group of women of Manaus-Brazil. Cervical samples were collected from 49 women, following the eligibility criteria of the study, and DNA was then extracted from the samples, which were analyzed for the presence of the virus in the genetic material through the polymerase chain reaction (PCR) using generic primers (GP05/06). Finally, identification of the viral subtypes was performed using specific primers for the detection of the main subtypes already examined (16 and 18). Positive HPV DNA was detected in 100% of the samples included in the study. Human papillomavirus 16 was the most prevalent subtype in the majority of lesions, accounting for 29 (59.2%) of the positive cases, and HPV 18 was detected in four (8.2%) women. In these 4 cases there was co-infection, with the presence of both HPV 18 and HPV 16. Therefore, 40.8% (20 cases) in which HPV DNA was detected presented infection with other subtypes of HPV not included in the study. This data has clinical implications related to cervical cancer prevention, as the current prophylactic HPV vaccines are only effective against high-risk HPV 16 and 18 subtypes.

Key Molecular Events in Cervical Cancer Development.

Cervical cancer is the fourth most common cancer among women. Infection by high-risk human papillomavirus (HPV) is the main aetiology for the development of cervical cancer. Infection by high-risk human papillomavirus (HPV) and the integration of the HPV genome into the host chromosome of cervical epithelial cells are key early events in the neoplastic progression of cervical lesions. The viral oncoproteins, mainly E6 and E7, are responsible for the initial changes in epithelial cells. The viral proteins inactivate two main tumour suppressor proteins, p53, and retinoblastoma (pRb). Inactivation of these host proteins disrupts both the DNA repair mechanisms and apoptosis, leading to rapid cell proliferation. Multiple genes involved in DNA repair, cell proliferation, growth factor activity, angiogenesis, as well as mitogenesis genes become highly expressed in cervical intraepithelial neoplasia (CIN) and cancer. This genomic instability encourages HPV-infected cells to progress towards invasive carcinoma. The key molecular events involved in cervical carcinogenesis will be discussed in this review.

LINC01101 and LINC00277 expression levels as novel factors in HPV-induced cervical neoplasia.

Recently long non-coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7-siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7-siRNA-transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV-induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV-positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV-induced cervical cancer, enriching our understanding of their potential role in this pathology.

Ten-year follow-up of human papillomavirus vaccine efficacy against the most stringent cervical neoplasia end-point-registry-based follow-up of three cohorts from randomized trials.

OBJECTIVE: Due to long lag time between infection/cancer diagnoses human papillomavirus (HPV) vaccination programs will deliver vaccine efficacy (VE) estimates against cancer end-points late. Cancer registry follow-up of population-based, randomised trial cohorts of vaccinated and unvaccinated women was undertaken for the estimation of VE against cervical intraepithelial neoplasia grade three and invasive cancer (CIN3+). METHODS: We report interim results with 98 561 person years of Finnish Cancer Registry -based follow-up of individually and/or cluster randomised cohorts of HPV-16/18 vaccinated and unvaccinated adolescent women enrolled in June 2003/2005, and between May 2004 and April 2005, respectively. The cohorts comprised 15 627 18- to 19-year-old unvaccinated women (NCT01393470), and 2 401 and 64 16- to 17-year-old HPV-16/18 vaccinated women participating the PATRICIA (NCT00122681) and HPV-012 (NCT00169494) trials, respectively. The age-aligned passive follow-up started 6 months after the clinical trials' end. RESULTS: During the follow-up of 4.5 to 10 years post enrolment we identified 75 cases of cervical intraepithelial neoplasia grade 3 (CIN3) and 4 cases of invasive cervical cancer (ICC) in the unvaccinated cohort, and 4 CIN3 cases in the HPV-16/18 vaccinated women. Diagnostic blocks were available for HPV typing from 87% of the cases. CIN3+ lesions were detectable in 54 cases. HPV16 was found in 26 of 50 unvaccinated CIN3+ cases, and in 3 CIN3+ cases in the HPV-16/18 vaccinated women. The latter were all baseline positive for cervical HPV16 DNA. Baseline data was not available for the unvaccinated women. Intention-to-treat VE against any CIN3+ was 66% (95% CI 8, 88). CONCLUSIONS: Ten years post vaccination the AS04-adjuvanted HPV-16/18 vaccine shows continued efficacy against CIN3+ irrespectively of HPV type. Vaccine efficacy was not observed in baseline HPV16 DNA positive subjects. TRIAL REGISTRATION NUMBER: NCT01393470.

HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions.

To clarify E1^E4's role during high-risk HPV infection, the E4 proteins of HPV16 and 18 were compared side by side using an isogenic keratinocyte differentiation model. While no effect on cell proliferation or viral genome copy number was observed during the early phase of either virus life cycle, time-course experiments showed that viral genome amplification and L1 expression were differently affected upon differentiation, with HPV16 showing a much clearer E4 dependency. Although E4 loss never completely abolished genome amplification, its more obvious contribution in HPV16 focused our efforts on 16E4. As previously suggested, in the context of the virus life cycle, 16E4s G2-arrest capability was found to contribute to both genome amplification success and L1 accumulation. Loss of 16E4 also lead to a reduced maintenance of ERK, JNK and p38MAPK activity throughout the genome amplifying cell layers, with 16E4 (but not 18E4) co-localizing precisely with activated cytoplasmic JNK in both wild type raft tissue, and HPV16-induced patient biopsy tissue. When 16E1 was co-expressed with E4, as occurs during genome amplification in vivo, the E1 replication helicase accumulated preferentially in the nucleus, and in transient replication assays, E4 stimulated viral genome amplification. Interestingly, a 16E1 mutant deficient in its regulatory phosphorylation sites no longer accumulated in the nucleus following E4 co-expression. E4-mediated stabilisation of 16E2 was also apparent, with E2 levels declining in organotypic raft culture when 16E4 was absent. These results suggest that 16E4-mediated enhancement of genome amplification involves its cell cycle inhibition and cellular kinase activation functions, with E4 modifying the activity and function of viral replication proteins including E1. These activities of 16E4, and the different kinase patterns seen here with HPV18, 31 and 45, may reflect natural differences in the biology and tropisms of these viruses, as well as differences in E4 function.

Immunoexpression of HPV 16/18 E6 and E7 oncoproteins in high-grade cervical squamous intraepithelial lesions in HIV-positive women.

The aim of this study was to assess the immunoexpression of human papillomavirus genotypes 16 and 18 (E6 and E7) oncoproteins in cervical high-grade squamous intraepithelial lesions (HSIL) of human immunodeficiency virus (HIV)-positive women. These results were also compared to the persistence and/or recurrence of lesions after loop electrosurgical excision procedure. Cervical samples from 158 patients were divided into three groups according to the presence or absence of HSIL in women who were or were not HIV-positive. By using the tissue microarray technique, immunohistochemistry was performed to analyze the expression of HPV 16/18 E6 and E7 oncoproteins. Cervical samples from 95 HIV-positive women and 63 HIV-negative women were studied. A statistically significant difference was found in the immunoexpression of E6 and E7 oncoproteins in samples from HIV-positive women with HSIL and that of women with non-neoplastic tissue (P < 0.001). There was also a statistically significant correlation between the immunoexpression of E6 (P = 0.012) and E7 (P < 0.001) oncoproteins in lesion persistence among HIV-positive women. Within the limitations of this study, the immunoexpression of HPV 16/18 E6 and E7 oncoproteins may have prognostic value regarding lesion persistence in HIV-positive women.

Disc large 1 expression is altered by human papillomavirus E6/E7 proteins in organotypic cultures of human keratinocytes.

Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions.

Prognostic implication of human papillomavirus types and species in cervical cancer patients undergoing primary treatment.

High-risk human papillomavirus (HPV) types are associated with cervical cancer. It is well established that individual HPV types vary in oncogenicity, but current data on their prognostic implication remain controversial. We examined the association between HPV types/species and the survival of 236 Chinese women aged 26-87 (mean 54.4) years after receiving primary treatment for cervical cancer. Overall, 45.8% were of FIGO stage I, 41.9% stage II, and 12.3% stage III. The four most prevalent types found were HPV-16 (60.2%), HPV-18 (21.6%), HPV-52 (11.9%), and HPV-58 (9.3%). Overall, 19.5% of patients had multiple-type infections, 78.4% harboured one or more alpha-9 species, and 28.8% harboured one or more alpha-7 species. After a median follow-up of 8.0 years, 156 (66.1%) patients survived. The 3-year overall survival rate was 75.5%. Factors independently associated with a poorer 3-year overall survival were age >60 years, tumour size >4 cm, lymph node involvement and treatment with radiotherapy+/-chemotherapy. Univariate analysis showed HPV-16 single-type infection was associated with a marginally poorer disease-specific survival (71.6% vs. 87.0%, HR: 1.71, 95% CI = 1.01-2.90), whereas non-HPV-16 alpha-9 species was associated with a better disease-specific survival (90.0% vs. 76.2%, HR: 0.36, 95% CI = 0.16-0.79). However, on multivariate analysis, HPV infection status irrespective of different grouping methods, including individual types, species, single-type or co-infection, did not carry any significant prognostic significance. In conclusion, we did not observe any association between infection with a particular HPV type/species and survival. An HPV type-based stratification in treatment and follow-up plan could not be recommended.

Robust HPV-18 production in organotypic cultures of primary human keratinocytes.

The productive program of the human papillomaviruses takes place in terminally differentiating squamous epithelia. In this chapter, we provide the protocols for robust production of HPV-18 in organotypic cultures of early passages of primary human keratinocytes. A critical step is the generation of genomic HPV plasmids in vivo by using Cre-loxP-mediated excisional recombination from a vector plasmid. We discuss the rationale for this approach. This system produces high yields of infectious virus and facilitates genetic analyses of HPV protein functions and their regulation in the context of recapitulated host tissue environment.

Sp100 provides intrinsic immunity against human papillomavirus infection.

UNLABELLED: Most DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA. IMPORTANCE: The intrinsic immune system provides a first-line defense against invading pathogens. Host cells contain nuclear bodies (ND10) that are important for antiviral defense, yet many DNA viruses localize here upon cell entry. However, viruses also disrupt, reorganize, and modify individual components of the bodies. In this study, we show that one of the ND10 components, Sp100, limits the infection of human skin cells by human papillomavirus (HPV). HPVs are important pathogens that cause many types of infection of the cutaneous and mucosal epithelium and are the causative agents of several human cancers. Understanding how host cells counteract HPV infection could provide insight into antimicrobial therapies that could limit initial infection.

Differential dependence on host cell glycosaminoglycans for infection of epithelial cells by high-risk HPV types.

Human papillomavirus (HPV) infection is the leading cause of cervical cancer world-wide. Here, we show that native HPV particles produced in a differentiated epithelium have developed different strategies to infect the host. Using biochemical inhibition assays and glycosaminoglycan (GAG)-negative cells, we show that of the four most common cancer-causing HPV types, HPV18, HPV31, and HPV45 are largely dependent on GAGs to initiate infection. In contrast, HPV16 can bind and enter through a GAG-independent mechanism. Infections of primary human keratinocytes, natural host cells for HPV infections, support our conclusions. Further, this renders the different virus types differentially susceptible to carrageenan, a microbicide targeting virus entry. Our data demonstrates that ordered maturation of papillomavirus particles in a differentiating epithelium may alter the virus entry mechanism. This study should facilitate a better understanding of the attachment and infection by the main oncogenic HPV types, and development of inhibitors of HPV infection.

The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

Chemically modified bovine beta-lactoglobulin inhibits human papillomavirus infection.

Previous studies have shown that 3-hydroxyphthalic anhydride-modified bovine beta-lactoglobulin is a promising anti-HIV microbicide candidate. Here we found that this chemically modified protein, designated JB01, exhibited highly potent antiviral activity against infection by human papillomaviruses (HPV), including HPV6, HPV16 and HPV18. Its anti-HPV activity was correlated with the percentage of modified lysine and arginine residues in JB01. This modified milk protein had no cytotoxicity at the concentration of 1 mg/ml, and it is highly stable at room temperature and 37 °C for at least 12 weeks. These results suggest that JB01 has good potential to be developed as an effective, safe and inexpensive antiviral agent for treatment and prevention of HPV infection.

Telomerase activity as an adjunct to high-risk human papillomavirus types 16 and 18 and cytology screening in cervical cancer.

Telomerase is a ribonucleoprotein comprising an RNA template, the telomerase-associated protein and its catalytic subunit, human telomerase reverse transcriptase (hTERT). Telomerase activation is a critical step in cellular immortalisation and development of cancer. Enhanced telomerase activity has been demonstrated in cervical cancer. In the present study telomerase activity and hTERT mRNA expression were evaluated and correlated with the presence of human papillomavirus (HPV) infection and cytological changes in the cervical lesions. Telomerase activity was assayed by telomeric repeat amplification protocol, hTERT mRNA expression by reverse transcriptase polymerase chain reaction and presence of high risk HPV (HR-HPV) infection by polymerase chain reaction. Out of 154 cervical samples of different cytology, 90 (58.44%) were positive for HR-HPV types 16/18, while among 55 normal cervical scrapes, 10 (18.18%) were HPV DNA positive. All 59 invasive cancer samples showed a very high telomerase activity. Among dysplasia, seven (63.6%) mild dysplasia, 18 (100%) of moderate, 20 (100%) of severe dysplasia and 6 (100%) carcinoma in situ (CIS) samples were positive with mild to moderate to high to very high telomerase activity respectively. Seven (12.7%) samples of apparently normal cervical scrapes were weakly positive for telomerase activity. We observed a good correlation (P<0.001) between telomerase activity and HR-HPV 16/18 positivity with a sensitivity of 88.1% for HPV and 100% for telomerase activity. It is suggested that telomerase activity may be used as an adjunct to cytology and HPV DNA testing in triaging women with cervical lesions.

Differences in human papillomavirus type may influence clinical outcome in early stage cervical cancer.

BACKGROUND: The presence of human papillomavirus (HPV), the HPV type and viral load in early stage cervical carcinoma were investigated in order to elucidate whether any of these factors were important for clinical outcome. PATIENTS AND METHODS: Twelve patients who were disease-free 5 years after diagnosis were matched and compared with 12 patients who died within 2 years. The presence of HPV, HPV type and viral load in their tumours was examined by PCR. RESULTS: The distribution and load of HPV was similar in the 2 patient groups. HPV-16 was, however, significantly more common in tumours of the surviving patients than in those of patients who died (88.9% and 18.2%, respectively, p = 0.0152). CONCLUSION: HPV-16 was significantly more common in early stage carcinomas of patients surviving more than 5 years in comparison to early stage carcinomas of patients with a poor prognosis.

[Effect of small interference RNA on E6, E7 mRNA of human papillomavirus type-18 in cervical cancer cells].

OBJECTIVE: To study the effect of small interference RNA on E6, E7 mRNA of human papillomavirus type-18 (HPV18) in cervical cancer cells. METHODS: The specific HPV18E6 and E7 siRNA synthesized by in virtro transcription. The cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay to determine the most suitable concentration, then cells were transfected into HPV18 cervical cancer cells by oligofectamine. HPV18E6, E7 mRNA level was examined by semi-quantitative RT-PCR, and the cell cycles were analyzed by flow cytometry. RESULTS: The activity of the HeLa cells had an obvious suppression after transfection to 0.57 +/- 0.05 and 0.62 +/- 0.04 compared with the contrast of 0.87 +/- 0.05. The expression of HPV18E6 mRNA level was 0.63 +/- 0.04 before transfection, decreased to 0.53 +/- 0.04, 0.46 +/- 0.02, 0.56 +/- 0.03 evidently after transfection at 24, 48, 72 hours. The expression of HPV18E7 mRNA level was 0.66 +/- 0.03 before transfection and decreased to 0.60 +/- 0.05, 0.52 +/- 0.04, 0.59 +/- 0.02 after transfection at 24, 48, 72 hours. The number of cells in G(2) phase was increased after HPV18E6 siRNA transfection from (1.4 +/- 1.2)% to (66.9 +/- 3.5)%, S phase was declined from (39.4 +/- 0.4)% to (0 +/- 5.5)%, and they were (47.2 +/- 0.5)% and (5.6 +/- 4.2)% in E7 group. CONCLUSION: RNAi exists in HeLa cells, and its effect has specificity.