Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.     

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus’ interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin’s stabilisation and formulation before its development as an alternative prophylactic means.     

人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。     

昆虫细胞偏爱密码子优化的HPV16L1基因在昆虫和哺乳动物细胞中的表达

目的获得有效表达人乳头瘤病毒16型（HPV16）L1基因的重组杆状病毒和腺病毒，为研究HPV的免疫保护机制提供材料。方法按照昆虫细胞密码子偏爱优化并合成HPV16LI基因，利用Bac—to-Bac昆虫表达系统获得表达HPV16L1基因的重组杆状病毒，利用AdEasy腺病毒载体系统获得表达HPV16L1基因的重组腺病毒载体。通过间接免疫荧光和Westernblot对HPV16L1基因表达进行鉴定，利用负染电子显微镜观察病毒样颗粒（VLP）的形成。结果获得了稳定表达HPV16L1蛋白的重组杆状病毒和重组腺病毒载体，在Sf9细胞和293细胞中可有效表达能被抗HPV16L1单克隆抗体识别的L1蛋白，分子质量单位为56ku，在Sf9细胞中可观察到VLP的形成。结论按照昆虫细胞密码子偏爱进行优化的HPV16L1基因，在昆虫细胞和哺乳动物细胞内均可有效表达。     

Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation

Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and approximately 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavirus (HPV) virions have greatly limited studies on many aspects of papillomavirus biology. To overcome these limitations, we developed a system to encapsidate the full-length papillomaviral genome into infectious virions, independently of viral DNA replication and epithelial differentiation. This transient-transfection-based system produces >1,000 times more infectious virus per cell culture dish than the much more labor-intensive organotypic culture. Furthermore, we show that this method allows the facile generation of infectious particles containing wild-type, mutant, or chimeric papillomaviral genomes, overcoming barriers to studying many facets of replication, host interactions, and vaccine and drug development, which has been limited by the insufficient availability of infectious virions.     

Inactivation of Polyomavirus SV40 as Surrogate for Human Papillomaviruses by Chemical Disinfectants

Human papillomaviruses (HPV) are non-enveloped DNA viruses infecting cutaneous and mucosal squamous epithelia. Sexually transmitted HPV-types that are carcinogenic to humans such as HPV16 can induce cervical and other anogenital cancers. Virus transmission through fomites such as inadequately disinfected gynecological equipment is a further potential transmission route. Since HPV cannot be easily grown in cell culture, polyomavirus SV40 has been used as a surrogate virus when testing the virucidal activity of chemical disinfectants. So far, studies that have compared the virucidal activity of different disinfectants against HPV and SV40 are lacking. Here, we evaluated the susceptibility of HPV16 pseudovirus and SV40 to seven active biocidal substances using quantitative suspension tests. Ethanol, glutaraldehyde (GTA), dodecyldipropylentriamin (DPTA), and ortho-phthalaldehydes (OPA) were able to reduce the infectivity of HPV16 pseudovirus >99.99% after 5 min. In contrast, isopropanol, peracetic acid (PAA), and quaternary ammonium compounds with alkylamines (QAC) only led to a slight or no reduction in infectivity. Concerning SV40, only GTA (60 min contact time), PAA, and OPA had virus-inactivating effects. In conclusion, the virucidal activity of three out of seven disinfectants tested was different for HPV16 pseudovirus and SV40. In this study, SV40 was shown to be a reliable surrogate virus for HPV when testing isopropanol-, GTA-, QAC-, and OPA-based disinfectants.     

HPV16 L1在整合型重组毕赤酵母中的表达及病毒样颗粒的纯化

目的 构建可稳定表达HPV16 L1的整合型重组毕赤酵母,并纯化自主组装成的HPV16 L1病毒样颗粒（VLPs）.方法 根据酵母密码子偏爱性优化HPV16 L1基因并克隆到pPIC3.5K表达载体,构建pPIC3.5K/HPV16 L1重组质粒;重组质粒经Bgl Ⅱ酶切线性化后,电转化至GS115菌株中,筛选HPV16 L1重组毕赤酵母.阳性整合菌株甲醇诱导后,以HPV16 L1单克隆抗体检测目的蛋白表达;采用肝素亲和层析法纯化HPV16 L1VLPs并进行透射电镜观察.结果 PCR、酶切和测序分析表明成功构建了pPIC3.5K/HPV16 L1重组质粒.成功构建的HPV16 L1重组毕赤酵母甲醇诱导后,Western blot证实重组酵母菌裂解产物存在HPV16 L1目的蛋白.肝素亲和纯化后,透射电镜观察到了直径大约55 nm的VLPs,其形态与HPV16天然病毒颗粒相似.结论 利用整合型重组毕赤酵母表达系统成功表达了HPV16L1蛋白,并用肝素亲和纯化可快速获得结构完整的HPV16 L1VLPs,为HPV16预防性疫苗的研制奠定基础.     

Antibody‐dependent and antibody‐independent uptake of HBsAg across human leucocyte subsets is similar between individuals with chronic hepatitis B virus infection and healthy donors

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero‐conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV‐infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti‐HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)‐dependent and Ab‐independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab‐dependent or Ab‐independent phagocytic HBsAg uptake may modulate presentation of HBsAg‐derived epitopes to antigen‐specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un‐complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un‐complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis‐related cellular functions are not altered in the context of CHB.     

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.     

A virus-based vaccine may prevent cervical cancer

High-risk human papillomaviruses (HPVs) are now recognized as the etiologic agents of invasive cervical cancer, a major cancer in women. A single HPV type (type 16) is responsible for about 50% of the cancers. The major capsid protein of papillomaviruses, L1, when expressed by recombinant DNA technology, has the intrinsic ability to assemble into virus-like particles (VLPs). In a recent study, a vaccine based on HPV 16 VLPs was tested in a placebo-controlled proof-of-principle trial in young women in the United States. The vaccine was found to prevent 100% of incident persistent HPV 16 infections and HPV 16-associated cervical intraepithelial neoplasia. These results offer promise that cervical cancer will be preventable by an HPV-based vaccine. Studies planned or in progress are examining the efficacy of the vaccine in men, in HIV-infected individuals, and in other parts of the world. Attempts are being made to prepare vaccines that can be administered more easily to large populations.     

高危型人乳头瘤病毒感染率及亚型分布与宫颈病变程度的相关性分析

目的探讨妇女高危型人乳头瘤病毒(HR-HPV)感染率及亚型分布与宫颈病变程度的相关性。方法回顾性分析2018年1月至2019年12月绍兴市3 307例妇女细胞学检查和HPV筛查结果。细胞学检查采用液基薄层细胞检测(TCT),HPV筛查采用聚合酶链式反应(PCR)-反向点杂交法,采用SPSS18.0软件进行统计学分析。结果3 307例受检妇女TCT结果未见上皮内病变细胞或恶性细胞(NILM)、非典型鳞状上皮细胞(ASC)和癌前病变(SIL)患者分别占90.84%、6.86%和2.30%。HPV阳性率19.11%,HR-HPV占82.52%。NILM、ASC和SIL患者HPV阳性率分别为14.38%、59.47%和85.53%,HR-HPV构成比分别为80.82%、83.33%和90.38%。HPV排前5的基因型为HPV52、HPV16、HPV58、HPV81和HPV53,但ASC和SIL患者最常见HPV16感染。HPV阳性患者重叠感染率为25.95%,SIL患者重叠感染率明显高于NILM和ASC患者(χ²=19.75和13.14,P <0.05)。HPV阳性率≥51岁年龄组明显高于其他所有年龄组,31~50岁年龄组明显高于≤30岁年龄组,差异均有统计学意义(χ²=24.23、29.31和5.90,P<0.05)。结论本地区SIL患者HPV阳性率、HR-HPV构成比和重叠感染率均明显高于NILM患者,HPV阳性率、HR-HPV构成比和重叠感染率与宫颈上皮内病变程度密切相关。随着年龄增大HPV阳性率呈上升趋势,应重视中老年妇女的宫颈癌筛查和防治教育。     

Vaccines against human papillomavirus and perspectives for the prevention and control of cervical cancer

Today, "persistent" infections by certain types of human papillomavirus (HPV) are considered necessary for developing cervical cancer. Producing efficient vaccines against these viruses may eventually lead to a great reduction in incidence and mortality rates of this cancer. In the case of HPV, the production of traditional vaccines usually based in dead or attenuated viruses is not possible due in part to the lack of systems where large quantities of viral particles could be obtained. Fortunately, the expression of the late L1 protein alone, or in combination with L2, leads to the generation of structures resembling true virions that have been called virus-like particles (VLPs) and constitute excellent candidates as prophylactic vaccines. VLPs have shown to be very immunogenic, and have prevented development of natural or challenged infections in both animal systems and humans. Recently, HPV16VLPs were shown to be very efficient to prevent the development of "persistent" infections, as determined by PCR assays, in a large group of vaccinated women. Therapeutic vaccines, on the other hand, are expected to have an impact on advanced lesions and residual illness, by taking advantage of the fact that early E6 and E7 genes are thought to be constitutively expressed in cervical tumors and precursor lesions. Finally, DNA-based vaccines could represent a useful alternative for preventing infections by genital HPV. This paper is available too at: http://www.insp.mx/salud/index.html.     

人乳头瘤病毒16型晚期基因L1的原核表达质粒的构建和鉴定

目的 构建人类乳头瘤病毒（ＨＰＶ）１６型晚期基因Ｌ１的原核表达质粒,以期从中选出能够高效表达ＨＰＶ１６Ｌ１的重组子,为进一步表达Ｌ１蛋白奠定基础。方法 应用定向克隆策略,将ＨＰＶ１６Ｌ１基因分别克隆到原核表达载体ｐＴｒａｃ９９Ａ和ｐＥＴ－２１ｃ中。结果 通过酶切鉴定选出了重组子ｐＴｒｃ９９Ａ－ＨＰＶ１６Ｌ１和ｐＥＴ－２１ｃ－ＨＰＶ１６Ｌ１。结论 ＨＰＶ１６Ｌ１的原核表达质粒构建成功。     

HPV 6b结构蛋白L1和沙眼衣原体CTL表位嵌合病毒样颗粒在昆虫细胞中的表达

目的为研制能同时防治人乳头瘤病毒(HPV)和沙眼衣原体(Ct)的嵌合疫苗。方法将Ct主要外膜蛋白(MOMP)插入到HPV衣壳蛋白L1羧基端,通过PCR、酶切、连接等方法构建了杆状病毒表达载体,进一步在大肠杆菌中组装成重组杆状病毒,经感染昆虫细胞表达嵌合病毒样颗粒,并经SDS-PAGE电泳和Westernblot分析鉴定,电镜负染。结果嵌合蛋白HPV6bL1/CtMOMP249-268在昆虫细胞中得到了表达,表达产物的相对分子质量为56000,与HPV6bL1单抗产生特异性反应,电子显微镜观察到病毒样颗粒形成。结论在昆虫细胞中表达的含HPV6bL1和CtCTL表位的嵌合蛋白,能自行组装成病毒样颗粒,为人乳头瘤病毒和沙眼衣原体嵌合疫苗的研究奠定基础。     

人乳头瘤病毒16型(新疆株)E6基因的原核表达

根据人乳头瘤病毒16（新疆株）E6基因编码序列设计引物，从含有HPV16 E6基因的质粒中扩增出含HPV16 E6基因的CNA片段，该片段全长450bp，将所得片段与pMD18-T载体连接，转化到JM109大肠杆菌中，筛选的阳性克隆扩增后，提取质粒DNA，用BamH I和Sal I酶切，回收450bp的目的片段，定向克隆到pET28a中，转化JM109受体菌，从JM109受体菌中提出质粒，再转化到BL21（DE3）中，筛选出转化体，用IPTG诱导表达，在PAGE上见到所表达的18kD的特异性的HPV16（新疆株）E6蛋白条带。     

人乳头瘤病毒58型E6E7融合基因重组巨细胞病毒的构建及鉴定

目的 利用人乳头瘤病毒(Human papillomavirus,HPV)58突变型E6E7(Mutant type E6E7,mE6E7)融合基因为靶点,巨细胞病毒株(Towne株)细菌人工染色体(SW102 Towne bacterial artificial chromosome,SW102-T-BAC)为载体,构建携带HPV58 mE6E7融合基因的重组病毒,探讨HPV58mE6E7的转化活性。方法 PCR扩增带有50 bp Towne 开放读码框(open reading frame,ORF)75左右同源臂的GalK、mE6E7及野生型E6E7(Wild type E6E7,wE6E7)融合基因片段,切胶回收纯化;将GalK片段电转到(SW102-T-BAC)感受态细胞,经过同源重组、GalK及氯霉素抗性筛选,获得SW102-T-ORF75-Galk-BAC克隆;然后分别将纯化的mE6E7及wE6E7电转到SW102-T-ORF75-Galk-BAC感受态细胞,经脱GalK及氯霉素抗性筛选,获得SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC克隆;提取所得克隆质粒DNA,转染ARPE-19细胞(以转染SW102-T-BAC的细胞及未转染细胞为对照),逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达状况;观察重组病毒T-ORF75-mE6E7及T-ORF75-wE6E7对转染ARPE-19细胞的影响,通过软琼脂克隆分析mE6E7及wE6E7转化活性。结果 获得了SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC的克隆。逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达正确。转染SW102-T-ORF75-wE6E7-BAC的细胞生长失去接触抑制,出现重叠生长现象,其形态由原来梭形变为变圆、肿胀、胞浆颗粒增多;并且在软琼脂中能够形成克隆;而转染SW102-T-ORF75-mE6E7-BAC、SW102-T-BAC及未转染细胞未出现上述细胞的特点。结论 获得了T-ORF75-mE6E7及T-ORF75-wE6E7重组病毒,并且重组病毒T-ORF75-mE6E7的转化活性已消除,为HPV58型治疗性疫苗的研究奠定了基础。     

Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins

SUMMARY. Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low-density lipoproteins. To determine whether the association is specific to low-density lipoproteins (LDL) or very low-density lipoproteins (VLDL), we fractionated HCV-containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low-density (VLDL) fraction. However, it could not be ruled out that virus-associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody-free virions. Very low-density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10–1.11 gml^-1 region of the gradients contained 60–70 nm particles. Particles with similar surface structure but having a diameter of only 30–40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti-HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino-terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.     

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.     

以人乳头瘤病毒16 L1为载体的流感通用疫苗初步研究

目的 构建以人乳头瘤病毒(human papillomavirus,HPV)16 L1为载体的甲型流感病毒M2基因胞外区(M2e)通用疫苗杆粒,利用昆虫细胞杆状病毒表达系统,进行初步的蛋白表达.方法 利用PCR技术将甲型流感病毒M2e基因序列与HPVl6 L1相连.经酶切、酶联将融合基因插入pFastBacHTA载体,在DH10Bac细胞中进行同源重组,经测序鉴定后构建M2e-HPV16 L1杆粒,脂质体转染sf9昆虫细胞,收获并扩增含有M2e-HPV16 L1的杆状病毒,经扩增后获得高效价的重组杆状病毒,用SDS-PAGE、免疫荧光法和电镜检测目的蛋白的表达.结果 构建了以HPV16 L1为载体的M2e通用疫苗杆粒,通过转染sf9昆虫细胞得到初步M2e-HPV16 L1融合蛋白表达.结论 成功构建了HPV16 L1与甲型流感病毒M2e融合蛋白的病毒样颗粒,为流感通用疫苗的研制奠定了基础.