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We unveiled the mechanism of how HPV employs Aurora kinase A (AurA) of host cells to exert its oncogenic capability synergistically. We systematically characterized the mode of interaction between E6-encoded by cancer-causing HPV and AurA.

Our results showed that while both COVID-19 and non-COVID-19 hospitalized patients differed in the composition, alpha and beta diversity, and metabolic potential of the naso-oropharyngeal microbiota compared with local controls, the microbial communities in the two hospitalized patient groups did not differ significantly from each other, indicating a limited impact of SARS-CoV-2 on the naso-oropharyngeal microbiota in hospitalized patients. Besides, we identified Alloprevotella and Solobacterium as bacterial genera uniquely enriched in COVID-19 patients, which may serve as more specific biomarkers for COVID-19 detection.

To investigate the interplay among the oral microbiota, HPV infection, traditional risk factors and patient outcomes in head and neck squamous cell carcinoma (HNSCC).A multi-center study of HNSCC patients with paired tumor and control tissues. We characterized the oral microbiota and HPV infection of tissues in 166 Chinese adults by sequencing the bacterial 16S rRNA V3-V4 and HPV L1 regions, respectively, and examined the associations among the oral microbiota, HPV and clinical features.A total of 15.7% of the surveyed HNSCC patients were positive for HPV DNA, with infection rates varying from 66.7% in oropharyngeal SCC to 10.4% in oral cavity SCC (OSCC). No HPV infection was detected in the surveyed hypopharyngeal SCC. HPV16 was largely the predominant type. HPV infection in non-OSCC, especially oropharyngeal SCC, was associated with advanced N stage and superior survival outcomes. Oral microbiota dysbiosis was observed in HNSCC tumors, with differentially abundant taxa mainly associated with HNSCC subtype, T stage, survival/relapse, HPV infection, and smoking. Notably, the enrichment of Fusobacterium in tumor tissues of OSCC patients was associated with no smoking, early T stage, early N stage, and better 3-year disease-specific survival.Our findings underscore the involvement of oral microbiota dysbiosis in OSCC pathogenesis, Fusobacterium is involved with improved OSCC patient outcomes, especially in patients lacking traditional risk factors. Understanding the complex interactions among the oral microbiota, HPV infection and other risk factors for HNSCC will provide important insights into the pathogenesis of HNSCC.

The human papillomavirus E6 and E7 oncoproteins interact with a different subset of host proteins, leading to dysregulation of the apoptotic, cell cycle, and signaling pathways. In this study, we identified, for the first time, that Aurora kinase B (AurB) is a bona fide interacting partner of E6. We systematically characterized the AurB-E6 complex formation and its consequences in carcinogenesis using a series of in vitro and cell-based assays. We also assessed the efficacy of Aurora kinase inhibitors in halting HPV-mediated carcinogenesis using in vitro and in vivo models. We showed that AurB activity was elevated in HPV-positive cells, and this correlated positively with the E6 protein level. E6 interacted directly with AurB in the nucleus or mitotic cells. A previously unidentified region of E6, located upstream of C-terminal E6-PBM, was important for AurB-E6 complex formation. AurB-E6 complex led to reduced AurB kinase activity. However, the AurB-E6 complex increased the hTERT protein level and its telomerase activity. On the other hand, AurB inhibition led to the inhibition of telomerase activity, cell proliferation, and tumor formation, even though this may occur in an HPV-independent manner. In summary, this study dissected the molecular mechanism of how E6 recruits AurB to induce cell immortalization and proliferation, leading to the eventual cancer development. Our findings revealed that the treatment of AZD1152 exerted a non-specific anti-tumor effect. Hence, a continuous effort to seek a specific and selective inhibitor that can halt HPV-mediated carcinogenesis should be warranted.

SARS-CoV-2 transcribes a set of subgenomic RNAs (sgRNAs) essential for the translation of structural and accessory proteins to sustain its life cycle. We applied RNA-seq on 375 respiratory samples from individual COVID-19 patients and revealed that the majority of the sgRNAs were canonical transcripts with N being the most abundant (36.2%), followed by S (11.6%), open reading frame 7a (ORF7a; 10.3%), M (8.4%), ORF3a (7.9%), ORF8 (6.0%), E (4.6%), ORF6 (2.5%), and ORF7b (0.3%); but ORF10 was not detected. The profile of most sgRNAs, except N, showed an independent association with viral load, time of specimen collection after onset, age of the patient, and S-614D/G variant with ORF7b and then ORF6 being the most sensitive to changes in these characteristics. Monitoring of 124 serial samples from 10 patients using sgRNA-specific real-time RT-PCR revealed a potential of adopting sgRNA as a marker of viral activity. Respiratory samples harboring a full set of canonical sgRNAs were mainly collected early within 1 to 2 weeks from onset, and most of the stool samples (90%) were negative for sgRNAs despite testing positive by diagnostic PCR targeting genomic RNA. ORF7b was the first to become undetectable and again being the most sensitive surrogate marker for a full set of canonical sgRNAs in clinical samples. The potential of using sgRNA to monitor viral activity and progression of SARS-CoV-2 infection, and hence as one of the objective indicators to triage patients for isolation and treatment should be considered.

Objectives: Little is known whether differences exist in virus shedding, immune and inflammatory response related to SARS-CoV-2 in people living with human immunodeficiency virus (PLWH). We assessed viral RNA and cytokine profiles of HIV and SARS-CoV-2 coinfection in Hong Kong. Methods: PLWH hospitalized with SARS-CoV-2 infection in Hong Kong were included, compared with age-matched and disease severity-matched SARS-CoV-2 infected controls (ratio of 1:5) from February 1st 2020 to July 31st 2020. SARS-CoV-2 infection was confirmed by public health laboratory and virus concentration was quantified by an in-house real-time reverse transcription-quantitative polymerase chain reaction. A panel of cytokines and chemokines were performed. Results: HIV patients had a similar respiratory shedding profile compared to controls. Duration of faecal shedding of patient A, B, C and D were at least 9, 10, 33, and 11 days, respectively. HIV patients had lower plasma levels of IL-10 and NT-pro-BNP. All 4 PLWH cases showed seroconversion to SARS-CoV-2 with anti-SARS-CoV-2 S antibodies detected in serum collected between day 18 and 30 after symptom onset. Conclusions: PLWH behaves similarly with HIV-negative controls in respiratory viral load, but with decrease in IL-10 and NT-proBNP. PLWH may have a lower risk of immunostimulatory effect due to lower IL-10.

We detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on disposable wooden chopsticks used by 5 consecutive asymptomatic and postsymptomatic patients admitted for isolation and care at our hospital. Although we did not assess virus viability, our findings may suggest potential for transmission through shared eating utensils.

Human papillomavirus (HPV) infection contributes to virtually all cases of cervical cancer, the fourth most common cancer affecting women worldwide. The oncogenicity of HPV is mainly attributable to the E6 and E7 oncoproteins. HPV-52 is the seventh most common HPV type globally, but it has a remarkably high prevalence in East Asia. In previous studies it has been speculated that the oncogenicity might vary among different HPV-52 variants. In the present study, we compared the oncogenicity of E6 derived from the HPV-52 prototype and three commonly found variants, V1 (K93R), V2 (E14D/V92L) and V3 (K93R/N122K), through molecular and phenotypic approaches. We demonstrated that cells containing V1 achieved higher colony formation and showed greater cell migration ability when compared to other variants, but no difference in cell immortalization ability was observed. At the molecular level, the three variants formed complexes with E6-associated protein (E6AP) and p53 as efficiently as the prototype. They degraded p53 and PSD95/Dlg/ZO-1(PDZ) proteins, including MAGI-1c and Dlg, to a similar extent. They also exhibited a similar subcellular localization, and shared a half-life of approximately 45 min. Our findings provide a clearer picture of HPV-52 E6 variant oncogenicity, which is important for further studies aiming to understand the unusually high prevalence of HPV-52 among cervical cancers in East Asia.

Simple Summary Among the over 200 human papillomavirus (HPV) genotypes identified, approximately 15 of them can cause human cancers. In this review, we provided an updated overview of the distribution of cancer-causing HPV genotypes by countries in East, Southeast and South Asia. Besides the standard screening and treatment methods employed in these regions, we unravel HPV detection methods and therapeutics utilised in certain countries that differ from other part of the world. The discrepancies may be partly due to health infrastructure, socio-economy and cultural diversities. Additionally, we highlighted the area lack of study, particularly on the oncogenicity of HPV genotype variants of high prevalence in these regions. Abstract Human papillomavirus (HPV) infection remains one of the most prominent cancer-causing DNA viruses, contributing to approximately 5% of human cancers. While association between HPV and cervical cancers has been well-established, evidence on the attribution of head and neck cancers (HNC) to HPV have been increasing in recent years. Among the cancer-causing HPV genotypes, HPV16 and 18 remain the major contributors to cancers across the globe. Nonetheless, the distribution of HPV genotypes in ethnically, geographically, and socio-economically diverse East, Southeast, and South Asia may differ from other parts of the world. In this review, we garner and provide updated insight into various aspects of HPV reported in recent years (2015–2021) in these regions. We included: (i) the HPV genotypes detected in normal cancers of the uterine cervix and head and neck, as well as the distribution of the HPV genotypes by geography and age groups; (ii) the laboratory diagnostic methods and treatment regimens used within these regions; and (iii) the oncogenic properties of HPV prototypes and their variants contributing to carcinogenesis. More importantly, we also unveil the similarities and discrepancies between these aspects, the areas lacking study, and the challenges faced in HPV studies.

In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.

Background Self-collected specimens have been advocated to avoid infectious exposure to healthcare workers. Self-induced sputum in those with a productive cough and saliva in those without a productive cough have been proposed, but sensitivity remains uncertain. Methods We performed a prospective study in 2 regional hospitals in Hong Kong. Results We prospectively examined 563 serial samples collected during the virus shedding periods of 50 patients: 150 deep throat saliva (DTS), 309 pooled-nasopharyngeal (NP) and throat swabs, and 104 sputum. Deep throat saliva had the lowest overall reverse-transcriptase polymerase chain reaction (RT-PCR)-positive rate (68.7% vs 89.4% [sputum] and 80.9% [pooled NP and throat swabs]) and the lowest viral ribonucleic acid (RNA) concentration (mean log copy/mL 3.54 vs 5.03 [sputum] and 4.63 [pooled NP and throat swabs]). Analyses with respect to time from symptom onset and severity also revealed similar results. Virus yields of DTS correlated with that of sputum (Pearson correlation index 0.76; 95% confidence interval, 0.62–0.86). We estimated that the overall false-negative rate of DTS could be as high as 31.3% and increased 2.7 times among patients without sputum. Conclusions Deep throat saliva produced the lowest viral RNA concentration and RT-PCR-positive rate compared with conventional respiratory specimens in all phases of illness. Self-collected sputum should be the choice for patients with sputum.

OBJECTIVE This study aimed to evaluate the incidence of and factors associated with persistence and clearance of oral human papillomavirus (HPV) infections. METHOD A prospective cohort study invited 458 subjects (231 HPV-positive and 227 HPV-negative at baseline) to attend follow-ups at 12 months. Those 231 HPV-positive subjects and 10 new infections were invited to reassessment at 24 months. We used next-gen sequencing for detection and genotyping of HPV. RESULTS α-HPV infections showed higher persistence rates than β/γ-HPV (22.7% vs 9.2% at 12 months [P

Epidemiological studies have revealed that a wild-type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of the virus’s greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of the AKT and K-Ras/ERK signaling pathways, thereby explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumors, all to a greater extent than the prototype HPV58 and other common variants., Human papillomavirus (HPV) type 58 is the third most commonly detected HPV type in cervical cancer among Eastern Asians. Our previous international epidemiological studies revealed that HPV58 carrying an E7 natural variant, T20I/G63S (designated V1), was associated with a higher risk of cervical cancer. We recently showed that V1 possesses a greater ability to immortalize and transform primary cells, as well as degrading pRB more effectively, than the prototype and other common variants. In this study, we performed a series of phenotypic and molecular assays using physiologically relevant in vitro and in vivo models to compare the oncogenicity of V1 with that of the prototype and other common natural variants. Through activation of the AKT and K-Ras/extracellular signal-regulated kinase (ERK) signaling pathways, V1 consistently showed greater oncogenicity than the prototype and other variants, as demonstrated by increased cell proliferation, migration, and invasion, as well as induction of larger tumors in athymic nude mice. This study complements our previous epidemiological and molecular observations pinpointing the higher oncogenicity of V1 than that of the prototype and all other common variants. Since V1 is more commonly found in eastern Asia, our report provides insight into the design of HPV screening assays and selection of components for HPV vaccines in this region. IMPORTANCE Epidemiological studies have revealed that a wild-type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of the virus’s greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of the AKT and K-Ras/ERK signaling pathways, thereby explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumors, all to a greater extent than the prototype HPV58 and other common variants.

From this population-based study of a Chinese general population, it was found that smoking, drinking, oral sex, and more sexual partners were associated with alpha human papillomavirus (HPV) infection of the oral cavity. Teeth brushing before sleep was protective for beta/gamma-HPVs., Background Knowledge of the prevalence of and risk factors for oral human papillomavirus (HPV) infection, especially cutaneous types, is limited. Methods A population-based study using next-generation sequencing consecutively recruited asymptomatic individuals aged 18–64 years from a proportional sampling of the general population of Hong Kong, according to age groups, gender, and regions of residence. We examined associations of alpha-, beta-, and gamma-HPVs from oral rinse samples with participants’ sociodemographics by logistic regression models. Results The prevalence of oral HPV infection among 1426 ethnic Chinese was 15.5% (95% confidence interval [CI], 13.7%–17.5%), 2.5% (95% CI, 1.8%–3.5%), 11.9% (95% CI, 10.3%–13.6%), and 2.9% (95% CI, 2.1%–3.9%) for any type, alpha-, beta-, and gamma-HPV, respectively. Prevalence of any high-risk HPV was 0.8% (95% CI, 0.4%–1.4%), and that of HPV-16 was 0.4% (95% CI, 0.2%–0.8%). HPV-8 and HPV-98 were the most common beta types detected, while HPV-4 and HPV-SD2R were the most common gamma types. Prevalence of alpha- and beta/gamma-HPV infection showed a similar pattern of increase with age, and was higher in men than women. Smoking, drinking, oral sex, and more sexual partners were associated with alpha-HPV. Teeth brushing before sleep was protective for beta/gamma-HPVs. Discussion The epidemiologic factors associated with oral infection with alpha-HPVs are different from those of beta/gamma-HPVs, suggesting different modes of acquisition and persistence.

This cross-sectional study estimates the prevalence of unidentified SARS-CoV-2 infection in the general population of Hong Kong after 3 major pandemic waves., Key Points Question What was the prevalence of unidentified SARS-CoV-2 infection in April 2021 after 3 major pandemic waves in Hong Kong, a city without complete lockdown? Findings In this cross-sectional study of 4198 participants of the general public in Hong Kong, 6 were identified as positive for anti-SARS-CoV-2 IgG after 3 major waves of COVID-19. The adjusted prevalence of unidentified infection was 0.15%, with fewer than 1.9 unidentified infections for every recorded case. Meaning The findings suggest that stringent isolation and quarantine policies even without complete city lockdown are successful in minimizing SARS-CoV transmission., Importance Seroprevalence studies inform the extent of infection and assist evaluation of mitigation strategies for the COVID-19 pandemic. Objective To estimate the prevalence of unidentified SARS-CoV-2 infection in the general population of Hong Kong. Design, Setting, and Participants A prospective cross-sectional study was conducted in Hong Kong after each major wave of the COVID-19 pandemic (April 21 to July 7, 2020; September 29 to November 23, 2020; and January 15 to April 18, 2021). Adults (age ≥18 years) who had not been diagnosed with COVID-19 were recruited during each period, and their sociodemographic information, symptoms, travel, contact, quarantine, and COVID-19 testing history were collected. Main Outcomes and Measures The main outcome was prevalence of SARS-CoV-2 infection. SARS-CoV-2 IgG antibodies were detected by an enzyme-linked immunosorbent assay based on spike (S1/S2) protein, followed by confirmation with a commercial electrochemiluminescence immunoassay based on the receptor binding domain of spike protein. Results The study enrolled 4198 participants (2539 [60%] female; median age, 50 years [IQR, 25 years]), including 903 (22%), 1046 (25%), and 2249 (53%) during April 21 to July 7, 2020; during September 29 to November 23, 2020; and during January 15 to April 18, 2021, respectively. The numbers of participants aged 18 to 39 years, 40 to 59 years, and 60 years or older were 1328 (32%), 1645 (39%), and 1225 (29%), respectively. Among the participants, 2444 (58%) stayed in Hong Kong since November 2019 and 2094 (50%) had negative SARS-CoV-2 RNA test results. Only 170 (4%) reported ever having contact with individuals with confirmed cases, and 5% had been isolated or quarantined. Most (2803 [67%]) did not recall any illnesses, whereas 737 (18%), 212 (5%), and 385 (9%) had experienced respiratory symptoms, gastrointestinal symptoms, or both, respectively, before testing. Six participants were confirmed to be positive for anti-SARS-CoV-2 IgG; the adjusted prevalence of unidentified infection was 0.15% (95% CI, 0.06%-0.32%). Extrapolating these findings to the whole population, there were fewer than 1.9 unidentified infections for every recorded confirmed case. The overall prevalence of SARS-CoV-2 infection in Hong Kong before the roll out of vaccination was less than 0.45%. Conclusions and Relevance In this cross-sectional study of participants from the general public in Hong Kong, the prevalence of unidentified SARS-CoV-2 infection was low after 3 major waves of the pandemic, suggesting the success of the pandemic mitigation by stringent isolation and quarantine policies even without complete city lockdown. More than 99.5% of the general population of Hong Kong remain naive to SARS-CoV-2, highlighting the urgent need to achieve high vaccine coverage.

Background Increasing evidence indicates an etiological role of human papillomavirus (HPV) in head and neck cancers, particularly oropharyngeal squamous cell carcinoma (OPSCC). However, the association between HPV and other cancers, including esophageal and tongue remains unclear. This study delineated the molecular characteristics of HPV18 E6 and E7 in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cell lines with reference to cervical cancer (HeLa). Methods We analysed the HPV transcription profiles of esophageal and tongue cancer cells through Next-generation RNA sequencing, and the role of HPV18 E6 and E7 in these cells was assessed via siRNA approach, Western blotting and immunofluorescence assays. Results Overall, the HPV transcription profiles of esophageal and tongue cancer cells mimicked that of cervical cancer cells, with notable disruption of E2, and expression of E6, spliced E6 (E6*), E7, E1 and L1 transcripts. As with cervical cancer cells, p53 and its downstream transactivation target, p21, were found to be the major targets of E6 in esophageal and tongue cancer cell lines. Intriguingly, E7 preferentially targeted p130 in the two esophageal cancer cell lines, instead of pRb as in cervical cancer. Tca83 exhibited an E7 to E6 transcript ratio comparable to HeLa (cervix), targeted the ERK1/2 and MMP2 pathways, and was dependent on E6 and E7 to survive and proliferate. In contrast, both the esophageal cancer cell lines were distinct from HeLa in these aspects. Conclusions This is the first study that delineates transcript expression and protein interaction of HPV18 E6 and E7 in esophageal and tongue cancer cell lines, suggesting that HPV plays a role in inducing these cancers, albeit via distinct pathways than those observed in cervical cancer.

Three highly pathogenic human coronaviruses can cause severe acute respiratory syndrome (SARS-CoV, SARS-CoV-2 and MERS-CoV). Although phylogenetic analyses have indicated ancient origin of human coronaviruses from animal relatives, their evolutionary history remains to be established. Using phylogenetics and "high order genomic structures" including trimer spectrums, codon usage and dinucleotide suppression, we observed distinct clustering of all human coronaviruses that formed phylogenetic clades with their closest animal relatives, indicating they have encompassed long evolutionary histories within specific ecological niches before jumping species barrier to infect humans. The close relationships between SARS-CoV and SARS-CoV-2 imply similar evolutionary origin. However, a lower Effective Codon Number (ENC) pattern and CpG dinucleotide suppression in SARS-CoV-2 genomes compared to SARS-CoV and MERS-CoV may imply a better host fitness, and thus their success in sustaining a pandemic. Characterization of coronavirus heterogeneity via complementary approaches enriches our understanding on the evolution and virus-host interaction of these emerging human pathogens while the underlying mechanistic basis in pathogenicity warrants further investigation.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host&ndash, microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P

The presence of a PDZ binding motif (PBM) in the human papillomavirus (HPV) E6 oncoprotein appears to be a characteristic marker of high oncogenic potential and confers interaction with a number of different cellular PDZ domain-containing substrates. The E6 PBM is also subject to phosphorylation, resulting in inhibition of E6 PDZ binding activity and instead allowing E6 to associate with 14-3-3 proteins. In this study, we analyzed the conditions under which the E6 PBM is phosphorylated. We demonstrate that in normal cycling cells, the levels of E6 phosphorylation are very low. However, following exposure of cells to oxidative stress or the induction of DNA damage, there is a striking increase in the levels of E6 phosphorylation. Depending on the specific stimulus, this phosphorylation of E6 can involve the ATM/ATR pathway and is performed primarily through Chk1, although the Chk2 pathway is also involved indirectly through activation of protein kinase A (PKA). To understand the biological relevance of these phospho-modifications of E6, we analyzed their effects upon the ability of E6 to inhibit p53 transcriptional activity. We show that an intact E6 phospho-acceptor site plays an essential role in the ability of E6 to inhibit p53 transcriptional activity on a subset of p53-responsive promoters in a manner that is independent of E6's ability to direct p53 degradation. These results are, to our knowledge, the first example of a DNA damage response controlling PBM-PDZ recognition. This study also provides links between the DNA damage response, the regulation of E6 PBM function, and the inhibition of p53 activity and begins to explain how HPV-infected cells remain within the cell cycle, despite activation of DNA damage response pathways during productive virus infections.IMPORTANCE The cancer-causing HPV E6 oncoproteins all possess a PDZ binding motif at their extreme carboxy termini. Depending upon whether this motif is phosphorylated, E6 can recognize PDZ domain-containing proteins or members of the 14-3-3 family of proteins. We show here that DNA damage response pathways directly signal to the E6 PBM, resulting in Chk1- and Chk2-driven phosphorylation. This phosphorylation is particularly pronounced following treatment of cells with a variety of different chemotherapeutic drugs. A direct functional consequence of this signaling is to confer an enhanced ability upon E6 to inhibit p53 transcriptional activity in a proteasome-independent but phosphorylation-dependent manner. These results are the first example of DNA damage signaling pathways regulating PBM-PDZ interactions and provide the mechanistic link between E6 PBM function and perturbation of p53 activity.

Human papillomavirus 58 (HPV58) is found in 10 to 18% of cervical cancers in East Asia but is rather uncommon elsewhere. The distribution and oncogenic potential of HPV58 variants appear to be heterogeneous, since the E7 T20I/G63S variant is more prevalent in East Asia and confers a 7- to 9-fold-higher risk of cervical precancer and cancer. However, the underlying genomic mechanisms that explain the geographic and carcinogenic diversity of HPV58 variants are still poorly understood. In this study, we used a combination of phylogenetic analyses and bioinformatics to investigate the deep evolutionary history of HPV58 complete genome variants. The initial splitting of HPV58 variants was estimated to occur 478,600 years ago (95% highest posterior density [HPD], 391,000 to 569,600 years ago). This divergence time is well within the era of speciation between Homo sapiens and Neanderthals/Denisovans and around three times longer than the modern Homo sapiens divergence times. The expansion of present-day variants in Eurasia could be the consequence of viral transmission from Neanderthals/Denisovans to non-African modern human populations through gene flow. A whole-genome sequence signature analysis identified 3 amino acid changes, 16 synonymous nucleotide changes, and a 12-bp insertion strongly associated with the E7 T20I/G63S variant that represents the A3 sublineage and carries higher carcinogenetic potential. Compared with the capsid proteins, the oncogenes E7 and E6 had increased substitution rates indicative of higher selection pressure. These data provide a comprehensive evolutionary history and genomic basis of HPV58 variants to assist further investigation of carcinogenic association and the development of diagnostic and therapeutic strategies. IMPORTANCE Papillomaviruses (PVs) are an ancient and heterogeneous group of double-stranded DNA viruses that preferentially infect the cutaneous and mucocutaneous epithelia of vertebrates. Persistent infection by specific oncogenic human papillomaviruses (HPVs), including HPV58, has been established as the primary cause of cervical cancer. In this work, we reveal the complex evolutionary history of HPV58 variants that explains the heterogeneity of oncogenic potential and geographic distribution. Our data suggest that HPV58 variants may have coevolved with archaic hominins and dispersed across the planet through host interbreeding and gene flow. Certain genes and codons of HPV58 variants representing higher carcinogenic potential and/or that are under positive selection may have important implications for viral host specificity, pathogenesis, and disease prevention.

Cervical cancer develops through the combined activities of the human papillomavirus (HPV) E6 and E7 oncoproteins. A defining characteristic of E6 oncoproteins derived from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at the extreme carboxy terminus of the protein which is absent from E6 proteins derived from the so-called low-risk HPV types. Within this PBM is also a protein kinase A (PKA) phospho-acceptor site, which is thought to negatively regulate the association of E6 with its PDZ domain-containing substrates. We can now show that phosphorylation of E6 by PKA and/or AKT confers the ability to interact with 14-3-3ζ. The interaction is direct and specific for the high-risk HPV E6 oncoproteins, although there are significant differences in the efficiencies with which HPV-16, HPV-18, and HPV-31 E6 oncoproteins can associate with 14-3-3ζ; this correlates directly with their respective susceptibilities to phosphorylation by PKA and/or AKT. We demonstrate here that the interaction between E6 and 14-3-3ζ also requires integrity of the E6 PBM, and downregulation of 14-3-3ζ results in a marked reduction in the levels of HPV-18 E6 expression in HeLa cells. Using phospho-specific anti-E6 antibodies, we also demonstrate significant levels of E6 phosphorylation in vivo . These studies redefine the potential relevance of the E6 PBM in the development of cervical cancer, suggesting that interaction with 14-3-3ζ, as well as the more well-established interactions with PDZ domain-containing substrates, is likely to be responsible for the biological activities attributed to this region of the high-risk HPV E6 oncoproteins.

Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

The human papillomavirus (HPV) E6 oncoprotein is fundamental to the ability of these viruses to induce human malignancy. A defining characteristic of the HPV E6 oncoproteins found in cancer-causing HPV types is the presence of a PDZ binding motif at their extreme C-terminus. Through this motif, E6 is able to interact with a large number of cellular proteins that contain PDZ domains. Many of these cellular proteins are involved in regulation of processes associated with the control of cell attachment, cell proliferation, cell polarity and cell signaling. How E6 targets multiple proteins containing the same recognition domain is still an open question. In this review, we highlight aspects of E6 function and biology that help to answer this question, and thereby provide insight into the role of these substrates during development of HPV-induced malignancy.

Problem statement: Recent studies using conventional Polymerase Chain Reaction have shown that anti-apoptotic (Cyclooxygenase-2, COX-2 and Nuclear Factor-κB, NF-κB) and proapoptotic mRNA (Bax and Bad) are involved in the tumorigenesis of colorectal cancer. The aim of this study was to localize the expression of anti and pro-apoptotic mRNA genes using Reverse Transcription in situ Polymerase Chain Reaction (RT in situ PCR) and immunodetection technique in the early stage of human colorectal adenocarcinoma. Approach: Reverse Transcription in situ Polymerase Chain Reaction (RT in situ PCR) and immunodetection technique was applied throughout of this studies. 20 paraffin-embedded tissue blocks of human colorectal adenocarcinoma samples was used compared to controls. Results: Morphologically, the glands and crypts were well-differentiated, enlarged and irregular with active secretion of mucin. COX-2, NF-κB, Bax and Bad mRNA were expressed in both normal and human colorectal cancer tissues. All mRNA genes were expressed in the cytoplasm and nuclei. However, COX-2 and NF-κB mRNA genes were highly expressed with higher intensity of brown staining compared to Bax and Bad at tubular epithelium cells. Conclusion: This study demonstrated that by using RT in situ PCR, COX-2 and NF-κB mRNA genes were shown to be involved in the development of human colorectal cancer.

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.