Activities of human papillomavirus 16 E6 natural variants in human keratinocytes

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.     

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.     

Distinctive distribution of HPV16 E6 D25E and E7 N29S intratypic Asian variants in Korean commercial sex workers

The distribution of HPV16 sequence variations differs geographically and specific HPV16 E6 and E7 variants might carry a high risk for development of invasive cervical cancer and cervical intraepithelial neoplasia in a given population. To investigate the genetic variation of HPV 16 E6 and E7 genes, genomic DNAs from 56 HPV16-infected commercial sex workers were extracted from their cervical swabs by using DNA isolation kit. The E6 and E7 coding region (34-880) with HPV16 E6/E7 specific PCR were amplified and analyzed by using the DNAstar software. At the nucleotide level, 26 variants of the HPV16 E6 and E7 genes were identified including 12 silent mutations. At the amino acid level, the isolates showed 14 variants including E6 Q14H, E6 D25E, E6 I27R, E6 H78Y, E6 L83V, and E7 N29S. The dominant HPV16 E6 and E7 variants were HPV16 E6 D25E (68%) and HPV16 E7 N29S (73%), respectively, which belong to Asian lineage. Although this study has some limitations such as a small sample size and not enough clinical data, these finding suggests that the distinctive distribution of HPV 16 As-variant E6 D25E and E7 N29S might be associated with geographical dependence rather than disease progression. Further study is needed to determine the clinical and biological effects of these variants.     

Genetic variability and functional implication of HPV16 from cervical intraepithelial neoplasia in Shanghai women

Human papillomavirus (HPV)16 gene mutation is usually associated with persistent HPV infection and cervical intraepithelial neoplasia (CIN). However, the functional implications of HPV16 mutations remain poorly understood.145 LCR/E6/E7 of the HPV16 isolates were amplified and sequenced, and HPV16 integration status was detected. In total, 89 SNPs (68 in the LCR, 13 in E6, 8 in E7) were discovered, 11 of which were nonsynonymous mutations (8 in E6, 3 in E7). The H85Y and E120D variants in E6 were significantly reduced in the high-grade squamous intraepithelial lesion (HSIL) group compared to the <HSIL group (P = .046 and .005), conversely the N29S in E7(P = .01). Amino acid substitutions (D32N/E, E36Q, H85Y, and E120D in E6 and N29H/S and R77C in E7) were predicted to have an effect on conserved structural and functional residues, and five amino acid substitutions (H85Y, E36Q, I34L, and D32E in E6; R77C in E7) would potentially change the secondary structure. “6329G>T,” a potential binding site for TATA-binding protein, is the most common in LCR variants. A4 (Asian) was associated with an increased risk of HSIL compared to A1–3(P = .009). The H85/E120 in E6 and N29 in HPV16 E7 might play a critical role in carcinogenesis by disrupting p53 and Rb degradation due to affecting their interaction, respectively. In a word, the findings in this study provide preventative and therapeutic interventions of HPV16 -related cervical lesions/cancer.     

新疆汉族、维吾尔族HPV16E6基因变异与子宫颈癌及宫颈病变的关系

目的探讨新疆地区汉族、维吾尔族宫颈癌及宫颈病变中HPV16型E6基因变异分布及频率,比较两个民族之间的差异。方法对140例HPV16阳性标本,设计E6基因特异性引物,通过PCR扩增HPV16E6全长基因,PCR产物直接测序,进行序列分析并与德国标准株对比,筛选突变位点。结果 123例汉族、维吾尔族妇女宫颈癌及宫颈病变标本中均存在HPV16E6变异株,其中突变率分别为:47.37%(27/57)和50%(33/66),E6突变位点主要是L83V、D25E、D64E、I73V、H78Y、D113E,其中L83V变异株在两个民族突变中占的比例均最高,汉族L83V突变频率为29.82%(17/57)显著低于维吾尔族的40.90%(27/66)(P0.05)。而汉族D25E突变频率为19.30%(11/57)显著高于维吾尔族的7.58%(5/66)(P0.05),D64E变异株在维族突变株中占的比例为6.1%,而汉族标本中未检出。结论两个民族HPV16 E6基因变异发生的位置和变异发生的频率存在差异,维吾尔族人群中HPV16E6基因D64E变异株可能与该地区维吾尔族宫颈癌高发存在一定的关系。     

Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.     

Keratin expression in equine normal epidermis and cutaneous papillomas using monoclonal antibodies.

Keratin expressions in normal equine epidermis and experimentally induced equine papillomas were studied by immunohistochemical methods with three different human cytokeratin monoclonal antibodies, 34 beta B4 (directed against component 1), 34 beta E12 (directed against components 1, 5, 10, 11) and 35 beta H11 (directed against component 8). Staining patterns with 34 beta B4 and 34 beta E12 in the normal equine epidermis did not differ from those in the normal human epidermis. In the early developing papilloma, keratinocytes showed an abnormal suprabasal staining pattern and expressed an additional 56 kD keratin protein detected by 34 beta E12. In the advanced papilloma, cytolytic cells in the outer spinous and the granular layers did not stain positively with any of the three antibodies used. In both early and advanced papillomas, the expression of high molecular weight keratin proteins, as detected by 34 beta B4 and 34 beta E12, did not correlate with the degree of keratinization. By electron microscopy, keratinocytes in the advanced papilloma showed a marked decrease of tonofibrils and desmosome-tonofilament complex. These alterations may result from an abnormality in both proliferation and functional terminal differentiation of keratinocytes in the papilloma. There were obvious differences in staining patterns with 35 beta H11 between the normal human and equine epidermis; 54 kD keratin protein was expressed in suprabasal layers of the equine normal and papillomatous epidermis. Thus, this keratin protein may be regarded as a "permanent" marker for the equine epidermis.     

Association between human papillomavirus 16 E6 variants and human leukocyte antigen class I polymorphism in cervical cancer of Swedish women

Persistent infection with human papillomavirus (HPV), particularly HPV16, represents the prime risk factor in cervical carcinogenesis. HPV variants (e.g., within the E6 gene) together with immunogenetic factors of the host may be responsible either for effective viral clearance, or alternatively, for viral persistence. Peripheral blood from 27 HPV16 positive Swedish women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for human leukocyte antigen (HLA) class I typing. Women with HLA-B*44, HLA-B*51, or HLA-B*57 who were infected with the HPV16 E6 variant L83V had an approximately four- to fivefold increased risk for cancer compared with controls (odds ratio [OR] = 3.5, 95% CI = 1.1-11.1, OR = 4.2, 95% CI = 1.19-14.69, or OR = 4.67, 95% CI = 1.2-18.6, respectively). Epitope predictive algorithm with SYFPEITHI revealed that the variant at amino acid 83 affects the binding affinity in association with HLA-B*44. Interestingly, the HLA-B*15 allele seems protective because it was absent in HPV16 positive cancer. It is concluded that specific HLA class I alleles, combined with certain HPV16 E6 variants, may be crucial for immune surveillance in cervical carcinogenesis. The evaluation of associations of HLA alleles with HPV variants may be helpful in defining prognostic markers and in designing vaccines capable of mediating immune protection against HPV infection.     

Differential Dimerization of Variants Linked to Enhanced S‐Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand‐Binding Domain

NR2E3 encodes the photoreceptor‐specific nuclear hormone receptor that acts as a repressor of cone‐specific gene expression in rod photoreceptors, and as an activator of several rod‐specific genes. Recessive variants located in the ligand‐binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive‐ (S‐) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S‐cones and non‐functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease‐causing LBD missense variants by bioluminescence resonance energy transfer (BRET²) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev‐erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.     

Key mutations in the spike protein of SARS-CoV-2 affecting neutralization resistance and viral internalization

To control the ongoing COVID-19 pandemic, a variety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been developed. However, the rapid mutations of SARS-CoV-2 spike (S) protein may reduce the protective efficacy of the existing vaccines which is mainly determined by the level of neutralizing antibodies targeting S. In this study, we screened prevalent S mutations and constructed 124 pseudotyped lentiviral particles carrying these mutants. We challenged these pseudoviruses with sera vaccinated by Sinovac CoronaVac and ZF2001 vaccines, two popular vaccines designed for the initial strain of SARS-CoV-2, and then systematically assessed the susceptivity of these SARS-CoV-2 variants to the immune sera of vaccines. As a result, 14 S mutants (H146Y, V320I + S477N, V382L, K444R, L455F + S477N, L452M + F486L, F486L, Y508H, P521R, A626S, S477N + S698L, A701V, S477N + T778I, E1144Q) were found to be significantly resistant to neutralization, indicating reduced protective efficacy of the vaccines against these SARS-CoV-2 variants. In addition, F486L and Y508H significantly enhanced the utilization of human angiotensin-converting enzyme 2, suggesting a potentially elevated infectivity of these two mutants. In conclusion, our results show that some prevalent S mutations of SARS-CoV-2 reduced the protective efficacy of current vaccines and enhance the infectivity of the virus, indicating the necessity of vaccine renewal and providing direction for the development of new vaccines.     

Keratin expression in cutaneous lichen planus

The characteristic expression of keratins by keratinocytes is well documented. A typical 'hyperproliferative' profile of epidermal keratin expression occurs in psoriasis, wound healing and warts. This study analyses keratin expression in cutaneous lichen planus to determine abnormalities of differentiation occuring in this inflammatory disorder. Using a panel of monoclonal antibodies 28 samples (20 patients) were studied. The results showed that squamous differentiation was unaffected, with keratins K1 and K10 being expressed normally for the site sampled. The main abnormalities included extension of reactivity of the basal cell marker, LH8, into the suprabasal compartment. Keratin K17, usually restricted to adnexal structures, was variably expressed in the basal and suprabasal layers of the interfollicular epithelium of affected epidermis. Keratins K6 and K16, found suprabasally in hyperproliferative states, were detected both basally and suprabasally in all diseased samples. The keratin profile in lichen planus is analogous to the wound healing response. Suprabasal keratin K17 is found in psoriasis, wound healing and viral warts so the changes in keratin K17 may reflect hyperproliferative changes. It is likely that the changes in epidermal keratin expression are due to up-regulation of specific keratin genes by the production of cytokines and inflammatory mediators from the lymphocytic infiltrate typical of lichen planus.     

High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors.     

The E6E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway

Non-melanoma skin cancer is the most frequent malignancy in Caucasian populations. Evidence suggests the involvement of cutaneous Human Papillomavirus (HPV) of the genus beta (beta) in this disease. The ability of E6 and E7 of mucosal HPV to promote cellular transformation and inhibit immune response-related pathways plays a key role in cervical carcinogenesis. beta HPV-38 E6 and E7 display transforming activities in in vitro and in vivo models, but their impact on immune surveillance is unknown. Here we show that HPV-38 E6 and E7 affect the IFN-induced up-regulation of MHC class I. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation is associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1. The down-regulation of these proteins is ultimately due to the inhibition of STAT-1 expression. Analysis of cells expressing either HPV-38 E6 or E7 suggests that these effects are primarily the result of E6 expression, although a contribution by E7 cannot be excluded. We conclude that HPV-38 encodes oncoproteins that potentially contribute to the evasion of host immune surveillance.     

Human papillomavirus 8 E6 disrupts terminal skin differentiation and prevents pro-Caspase-14 cleavage

Expression of the betapapillomavirus (betaPV) E6/E7 genes has been shown to impair both keratinocyte differentiation and apoptosis. Especially late-terminal keratinocyte differentiation shares certain aspects with apoptosis, such as fragmentation of DNA and activation of caspases. Here we investigated the disruption of keratinocyte differentiation in organotypic skin (raft) cultures of primary (PHK) and immortalized (N/TERT) human keratinocytes, in particular by human papillomavirus (HPV)8.Immunohistochemical analysis of HPV5 and HPV8 E6/E7-expressing PHK revealed thickening of the rafts and complete absence of stratum corneum formation, even after 18 days of culture. This phenotype was confirmed in N/TERT raft cultures. When expressed separately, the aberrant morphology was observed only in rafts expressing E6, not E7. Immunofluorescence analysis of HPV8 E6 PHK rafts showed an increase in number and size of Filaggrin- and Caspase-14-positive cells in the granular layer. In raft lysates analyzed by western-blot, the presence of pro-Caspase-14 in the differentiated keratinocytes was confirmed, but in the HPV8 E6 rafts none of the Caspase-14 subunits were detected.In conclusion, in the raft system, HPV8 E6 prevented late-terminal keratinocyte differentiation resulting in an accumulation of Filaggrin and pro-Caspase-14-positive cells in the absence of stratification. This differentiation arrest was accompanied by the failure to express Caspase-14 subunits, suggesting absence of Caspase-14 activation and probable abrogation of Filaggrin maturation in HPV8 E6-expressing keratinocytes.     

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.     

Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.     

A novel real-time genotyping assay for detection of the E6-350G HPV 16 variant

It has been suggested that some E6 human papillomavirus (HPV) type 16 variants could be involved in viral persistence and progression of HPV infection. A novel one-step allelic discrimination real-time PCR was evaluated for E6-350G variant detection in 102 endocervical HPV 16 positive samples. This assay was also used to assess the distribution of this variant in Spanish women with cervical cancer related to HPV 16.The detection limit for the allelic discrimination assay was 50 copies per reaction, even where the E6-350G variant represents only 20% of the variants in the sample. Complete concordance was observed between DNA sequencing and the novel AD RT-PCR assay. Fourteen E6-350T reference strains and 18 E6-350G variants were detected out of 32 endocervical samples from women with cervical cancer. The average age of women who were infected by the E6-350G HPV 16 variant was 10 years lower in these samples than in women who were infected by the reference strain.This novel allelic discrimination assay is a fast, sensitive and specific method for detection of the E6-350G HPV 16 variant.     

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.     

N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel

Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.