Expression Pattern and Subcellular Localization of Human Papillomavirus Minor Capsid Protein L2

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18⁺ cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18⁺ high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18⁺ cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.     

Increased expression of cellular RNA‐binding proteins in HPV‐induced neoplasia and cervical cancer

The expression profile of a panel of RNA‐binding proteins (heterogeneous ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, hnRNP H, hnRNP I, ASF/SF2, SR proteins, HuR and U2AF⁶⁵) and markers of differentiation, proliferation and neoplasia (cytokeratin (CK) 13, CK‐14, proliferating cell nuclear antigen (PCNA), Syndecan‐1 and p16INK4a) were analyzed in 50 formalin fixed paraffin embedded cervical tissues using immunohistochemistry. The samples included histologically normal cervical epithelium, human papillomavirus (HPV) induced low‐grade and high‐grade pre‐malignant lesions and cervical cancers. All samples were tested for HPV DNA using nested PCR. Forty‐nine of the 50 tissue samples tested positive for HPV, 27 tissue samples (54%) were HPV‐16 positive and 4 samples (8%) were HPV‐18 positive. The immunohistochemistry results detected different expression levels of the various proteins in basal epithelial cells in histologically normal epithelium followed by an increase in expression in the intermediate layers, whereas the superficial layers remained negative for all tested RNA‐binding proteins. Expression of all RNA‐binding proteins increased in neoplastic lesions and highest expression was detected in cervical cancers. p16INK4a had a stronger association with high‐grade lesions when compared with the RNA‐binding proteins. The expression profile of the RNA‐binding proteins is similar to PCNA expression in histologically normal epithelium as well as in lesions (low‐grade and high‐grade) and cervical cancers. As PCNA expression has been suggested to mimic HPV E6/E7 expression in cervical epithelium, the results suggest the RNA‐binding protein analyzed here regulate HPV early gene expression directly and late gene expression indirectly. J. Med. Virol. 81:897–907, 2009. © 2009 Wiley‐Liss, Inc.     

L1-Specific Protection from Tumor Challenge Elicited by HPV16 Virus-like Particles

A single injection of HPV16 L1 virus-like particles induced potent CD8-mediated protection from tumor challenge by C3 cells, a line derived from embryonic mouse cells transfected with the HPV16 genome. L1 RNA, but not protein, was detected biochemically in C3 cells. These results indicate that low-level expression of HPV16 L1 can occur in proliferating cells and serve as a tumor vaccine target. Although L1 expression is generally thought to be restricted to terminally differentiated epithelial cells, these results suggest that additional analysis for low-level L1 expression in proliferating cells of HPV-induced lesions is warranted and might help in predicting the clinical potential of HPV L1 virus-like particle-based vaccines.     

Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology

Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women.Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv^® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens.Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004)Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is strongly associated with high-grade histopathology diagnoses. The multiplicity of HPV infections may have an important role in high-grade histopathology diagnoses (≥CIN3) in HPV L1-positive cases.     

子宫颈病变组织中低分子量蛋白酶体表达与HPV16感染的关系

目的观察低分子量蛋白酶体(low molecular-weight protein,LMP)在子宫颈病变组织中的mRNA和蛋白表达,探讨其与HPV16感染的关系。方法以152例新疆维吾尔族妇女正常子宫颈上皮、子宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)和子宫颈鳞癌(cervical squamous cell carcinoma,CSCC)患者为研究对象,采用RT-PCR和免疫组化法鉴定LMP2和LMP7mRNA及蛋白表达水平;采用PCR技术检测相应标本HPV16感染情况。结果 (1)LMP2、LMP7随着子宫颈病变的加重其蛋白表达逐渐降低,且mRNA表达水平与蛋白表达趋势相一致。在CIN中LMP2、LMP7蛋白表达下调和缺失率分别为25.0%/15.6%、29.7%/23.4%;在子宫颈癌中LMP2、LMP7蛋白的表达下调和缺失率分别为17.5%/34.9%、23.8%/41.3%。临床病理参数进行分析发现LMP2、LMP7与子宫颈癌分化程度及淋巴结转移密切相关(P<0.05)。(2)PCR结果显示,HPV16的检出率随着子宫颈病变的进展而增加,在慢性子宫颈炎、CIN和子宫颈癌组织中阳性率分别为8%(2/25)、67.2%(43/64)和77.8%(49/63),且随着肿瘤恶性程度的增加其阳性表达率增加,各组间阳性表达差异有统计学意义(P<0.05)。在CIN中LMP7表达下调与HPV16感染有关(P<0.05),子宫颈癌中LMP2和LMP7表达下调与HPV16感染有关(P<0.05)。结论 LMP基因的转录表达下调或蛋白质表达缺失与维吾尔族妇女子宫颈癌病变进程密切相关,其中HPV16感染可能是重要原因之一。     

宫颈癌组织APOBEC3A蛋白的表达与高危型HPV感染的相关性研究

 目的： 探讨宫颈癌组织中载脂蛋白 B mRNA 编辑酶催化多肽样蛋白3A（APOBEC3A）表达与高危型人乳头瘤病毒（HPV）感染的关系。方法： 采用免疫组化法检测26例宫颈癌、27例宫颈上皮内瘤变（CIN）I～III和22例正常宫颈组织APOBEC3A蛋白的表达,同时使用凯普分型检测试剂盒对3组样品分别进行高危HPV16/HPV18分型检测。以脂质体法转染APOBEC3A质粒进入HeLa细胞,RT-qPCR与Western blotting验证APOBEC3A对高危型HPV18 E6 mRNA以及蛋白表达的影响。结果：宫颈癌组织、CIN组织以及正常宫颈组织中APOBEC3A蛋白表达的阳性率分别为46.2%、92.6%和86.4%,宫颈癌组织中APOBEC3A蛋白表达较正常宫颈组织明显下降（P<0.01）。宫颈癌组织、CIN及正常宫颈组织中HPV16感染阳性率分别为92.3%、77.8%和54.5%; HPV18感染阳性率分别为80.8%、51.8%和68.2%;APOBEC3A蛋白表达与HPV18感染阳性率呈负相关(P<0.05)。增加HeLa细胞中APOBEC3A的表达明显降低了HPV18 E6 mRNA以及蛋白的表达。结论：在宫颈癌组织中APOBEC3A高表达可以对抗HPV18感染,并抑制HPV18 E6的转录和表达。     

人乳头瘤病毒16型L1/E7融合蛋白在原核细胞中的表达及免疫效果观察

目的构建能表达L1E7融合蛋白的原核表达菌株,纯化蛋白,并观察其免疫效果。方法用PCR方法分别扩增出C末端部分缺失的HPV16L1基因和HPV16E7编码基因N端部分序列。将上述基因连接,构建融合基因L1ΔCE7N并将其插到原核表达载体pGEX-2T中进行融合蛋白表达纯化,然后观察其免疫效果。结果L1ΔCE7N融合基因测序结果表明,序列与设计相符,读码框架正确。将其插入原核表达质粒在大肠埃希菌中获得高效表达;经Wester-Blot鉴定在相对分子质量约85×103处有特异性表达带,与预期相符。用亲和层析和分子筛可纯化L1ΔCE7N融合蛋白,将其免疫C57BL/6小鼠,结果表明融合蛋白能诱发高滴度L1、E7抗体,并能保护小鼠免受TC-1肿瘤细胞的攻击。结论本实验在原核系统中高效表达并纯化了L1ΔCE7N融合蛋白,该蛋白可作为预防和治疗HPV16感染以及相关肿瘤的候选疫苗株。为研制HPV16预防治疗性疫苗探索一条经济、易普及的途径。     

临床宫颈组织标本HPV16 L1蛋白检测

 目的 检测HPV16阳性宫颈标本L1蛋白的表达,发掘宫颈损伤程度与L1蛋白表达的规律。方法 PCR法对宫颈炎、宫颈上皮内瘤变（CINⅠ~Ⅱ）、原位癌、早期浸润癌及中晚期癌标本进行型别检测,ELISA法及免疫组化法检测HPV16阳性标本中HPV L1蛋白表达。结果 54例宫颈组织中HPV16阳性为46例（85.2%）。随着宫颈损伤加重,HPV16阳性标本的抗原抗体反应逐渐减弱;免疫组化阳性反应仅出现于上皮组织内,反应强度及分布与宫颈损伤程度及癌变组织的分化状态有关。结论 HPV16 L1蛋白的表达同时受宫颈组织损伤程度及宿主细胞分化状态的影响。早期对HPV L1蛋白进行检测,可为宫颈癌的早期预防及临床诊治提供理论指导。     

悬浮培养昆虫细胞表达重组HPV16 L1蛋白

目的 用悬浮培养昆虫细胞方式表达人乳头瘤病毒16型(HPV16)L1蛋白,为获得病毒样颗粒(VLPs)及进一步深入研究疫苗和诊断试剂盒打基础.方法 优化昆虫细胞Sf9的悬浮培养条件;优化扩增病毒和蛋白的条件;空斑试验测定病毒滴度;SDS-PAGE和Western Blot分析目的蛋白的表达情况;透射电镜观察细胞内HPV16 L1蛋白形成的VLPs.结果 优化后,悬浮培养昆虫细胞初始接种密度为5×105cell/ml,以MOI(Multiplicity ofInfection)=10接种重组病毒rBacV/HPV16 L1,72～84h收获细胞沉淀为最佳.经透射电镜观察,在重组病毒感染的昆虫细胞内,存在HPV16L1蛋白形成的VLPs.结论 优化了细胞悬浮培养、病毒扩增和蛋白表达的条件;电镜观察在重组病毒rBacV/HPV16L1感染的昆虫细胞中,HPV16 L1蛋白可形成VLPs.     

Quantitative human papillomavirus 16 and 18 levels in incident infections and cervical lesion development

Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV‐16 and HPV‐18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990 to 2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri‐annually, and women were referred for colposcopically directed biopsy when indicated. Quantitative real‐time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV‐16 and HPV‐18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV‐16 mRNA levels at the time of incident HPV‐16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2–3 (HR per 1 log₁₀ increase in mRNA = 6.36, 95% CI = 2.00–20.23). Increasing HPV‐16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV‐16 E7 DNA levels nor HPV‐18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR] = 10.05, 95% CI = 1.09–92.56) and increased HPV DNA (VLR = 16.80, 95% CI = 1.46–193.01). In newly detected HPV‐16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre‐cancer. J. Med. Virol. 81:713–721, 2009 © 2009 Wiley‐Liss, Inc.     

Inversed Expression Patterns of S100A4 and E‐Cadherin in Cervical Cancers: Implication in Epithelial–Mesenchymal Transition

Cervical cancer/CC is the third commonest female malignancy worldwide. The aggressive growth and distal metastases are the leading causes of CC mortality, which is largely due to epithelial–mesenchymal transition/EMT. Fibroblast specific protein S100A4 promotes cancer metastasis and epithelial type cadherin/E‐cadherin play pivotal roles in cell–cell and cell–extracellular matrix interaction. Therefore, the expression patterns of S100A4 and E‐cadherin reflect statuses of EMT of carcinoma cells. However, S100A4 expression and its relevance with E‐cadherin and HPV16 infection in cervical cancers remain unknown. This study aims to address the above issues using cervical cancer specimens. Immunohistochemistry reveals that the levels of mesenchymal marker S100A4 is upregulated (>++) in cervical adenocarcinomas/CACs (12/16; 75%) and squamous cell carcinomas/CSCCs (23/28; 82%) than that in noncancerous glandular epithelia/GE (0/12; 0%) and squamous epithelia/SE (0/12; 0%). Epithelial marker membranous E‐cadherin is remarkably reduced on the surface of CAC and CSCC cells (P = 0.00; P = 0.00), especially those showing poorly differentiated phenotypes (P < 0.05) in comparison with their noncancerous counterparts. Correlative analyses revealed an inverse relationship between S100A4 and E‐cadherin expression among the cervical cancer samples (P = 0.01, r = –0.38). S100A4 expression level in HPV16‐infected group is higher than that in HPV16‐free group (P = 0.02). These results suggest the close correlation of S100A4 upregulation with cervical cancer formation and HPV16 infection and E‐cadherin reduction with the grades of CC dedifferentiation. The concurrent gain of S100A4 and loss of membrane E‐cadherin suggest EMT tendency of CC cells and can be regarded as an unfavorable prognostic parameter of CC patients. Anat Rec, 300:2184–2191, 2017. © 2017 Wiley Periodicals, Inc.     

Phenotypic effects of HPV-16 E2 protein expression in human keratinocytes

Expression of the HPV E2 open reading frame in cervical cancer cells has been shown to affect the expression of both viral and cellular genes. We have examined the phenotypic effects of the expression of human papillomavirus 16 E2 open reading frame in the human keratinocyte cell line HaCaT. Increased levels of apoptotic cell death were seen within 24 h of the transfection of HPV-16 E2 expression constructs. However, in those cells which survived selection and retained the intact E2 ORF, long-term stable expression of E2, as detected by RT-PCR, produced cells which developed phenotypes typical of terminally differentiated cells. These included characteristic morphological changes and expression of involucrin, filaggrin and senescence markers. This provides the first evidence of a role for E2 in stimulation of the normal epithelial differentiation programme, which would promote the progression of the HPV life cycle.     

Arginase is involved in cervical lesions progression and severity

BackgroundLittle is known about the relationship between arginase, an immunosuppressive enzyme, and cervical lesions. The present study is aimed at evaluating arginase activity in plasma and mRNA arginase isoforms expression in cervical cells of patients with abnormal cytology and identifying their relationship with Human papillomavirus (HPV) related parameters such as: HPV type, HPV circulating viral load and anti-HPV16 IgG.MethodsThis study included 77 women with cervical lesions and 95 matched controls. Arginase activity was detected by colorimetric assay. Arginase mRNA expression and HPV viral load were evaluated by quantitative real time PCR and anti-HPV16 antibodies were assessed by ELISA.ResultsCompared to controls, the arginase activity was higher among women with cervicitis / low grade squamous intraepithelial lesions (LSIL) (OR: 1.872, 95% CI: 0.833–4.210), and also among women with high-grade squamous intraepithelial lesions (HSIL) / squamous cell carcinoma (SCC) (OR: 3.358, 95% CI: 1.670–8.910). Compared to controls, mRNA expression was significantly upregulated in women with cervical cervicitis and SIL for ARG1, and in women with cancer lesions for ARG2. Arginase activity was positively correlated to ARG2 mRNA expression but not to ARG1. High arginase activity was associated with HPV16, high levels of HPV viral load, and low levels of anti-HPV16 antibodies.ConclusionsOur findings demonstrated that arginase activity and isoforms expression were enhanced in women with HPV-related precancerous lesions and cervical cancer. Further studies are needed to identify how arginase enzyme induces disease progression and severity.     

Effects of Fungi and Eosinophils on Mucin Gene Expression in Rhinovirus-Infected Nasal Epithelial Cells

Purpose

Fungi, rhinoviruses (RVs), and eosinophils are associated with upper respiratory diseases. We evaluated the effects of fungal stimulation and eosinophil co-culture on the expression of mucin genes in RV-infected nasal polyp epithelial cells.

Methods

Nasal polyp epithelial cells were obtained from chronic rhinosinusitis patients. Cultured epithelial cells were stimulated with Alternaria and Aspergillus with or without RV-16 infection. The epithelial cells were co-cultured with eosinophils for 16 h. MUC4, MUC5AC, MUC5B, and MUC8 mRNA expressions in the epithelial cells were quantified using real-time RT-PCR. To determine the underlying mechanism, nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and mitogen-activated protein kinase (MAPK) inhibitors were used to inhibit mucin gene expression.

Results

Fungi and RV-16 induced mucin gene expression in nasal polyp epithelial cells. However, there was no synergistic increase in mucin gene expression, with the exception of MUC4 mRNA expression stimulated by 25 µg/mL Aspergillus. When RV-16-infected epithelial cells were stimulated with fungi and then co-cultured with eosinophils, MUC4, MUC5B, and MUC8 mRNA expressions increased. Mucin gene expression was inhibited by NF-κB inhibitors.

Conclusions

RV-16, airborne fungi, and eosinophils may exacerbate the inflammatory process in nasal mucosal diseases by enhancing mucin gene expression.     

人乳头瘤病毒6b L1/16E7嵌合蛋白的基因克隆和表达

目的 研究HPV6bL1/16E7嵌合蛋白的基因克隆及其在昆虫细胞的表达,为防治尖锐湿疣和宫颈癌的基因工程疫苗研究作准备。方法 用PCR扩增出HPV6bL1/16E7嵌合蛋白基因,将其克隆到杆状病毒转移载体pVL1393,制备重组杆状病毒并感染昆虫细胞表达HPV6bL1/16E7嵌合蛋白。结果 HPV6bL1/16E7嵌合蛋白基因在昆虫细胞中得到了表达,并可自组装形成病毒样颗粒。结果 昆虫细胞表达的HPV6bL1/16E7嵌合病毒样颗粒可进一步用于HPV感染的免疫机理及基因工程疫苗研究。