黄病毒属病毒复制子载体研究进展

RNA复制子是一种能自主复制的RNA载体,保留了病毒非结构蛋白(复制/转录酶)基因,而结构蛋白基因缺失或由外源抗原基因替代,复制/转录酶可控制载体RNA在细胞质中高水平复制以及外源基因的高水平表达。在黄病毒属病毒感染性克隆基础上,其复制子载体得到了成功的构建。黄病毒属病毒复制子为病毒基因组结构功能研究、表达载体构建、假病毒包装及新型疫苗制备等提供了新的技术平台。本文综述黄病毒属病毒复制子的构建原理、方法及应用。     

人肠道病毒D68型微复制子的建立

建立人肠道病毒D68型(EV-D68)微复制子体系。利用增强绿色荧光蛋白(EGFP)或萤火虫荧光素酶(FLuc)报告基因替换病毒编码区,通过酶切连接,构建EV-D68 T7和PolⅠ系统微复制子体系,获得重组质粒pT7-miniEGFP、pT7-miniFLuc、pHH21-miniEGFP、pHH21-miniFLuc和pHH21-miniFLuc_(ΔpolyC)。微复制子转染RD细胞,48h后荧光显微镜观察EGFP表达情况或用双报告系统检测荧光素酶表达水平。T7和PolⅠ系统微复制子均可表达报告基因,但PolⅠ系统荧光素酶表达水平是T7系统的5倍。并且病毒非编码区的PolyC区域对于病毒蛋白的表达起着重要的作用。本研究成功构建了EV-D68微复制子体系,为EV-D68非编码区功能研究提供工具。     

表达EGFP报告基因口蹄疫病毒亚基因组复制子的构建

【目的】构建含有EGFP报告基因的口蹄疫病毒(FMDV)亚基因组复制子系统。【方法】利用融合PCR方法,将EGFP报告基因替换O型FMDV全长c DNA克隆中的前导蛋白Lb和结构蛋白P1基因,构建含有EGFP报告基因的FMDV亚基因组复制子FMDV-EGFP。复制子质粒连续转化、测序检验复制子载体的稳定性。Not I线性化的复制子FMDV-EGFP用脂质体介导法转染表达T7 RNA聚合酶的BSR/T7细胞后,不同时间段观察EGFP荧光表达情况。转染的细胞用流式、间接免疫荧光、RT-PCR和Western blot检测该复制子载体的自主复制能力和口蹄疫病毒蛋白的表达情况。【结果】复制子质粒的连续转化及测序表明报告基因可以稳定存在。FMDV-EGFP复制子转染BSR/T7细胞3 h后在荧光显微镜下能够看到绿色荧光,EGFP荧光信号随着转染时间的延长逐渐增加,并且荧光信号可持续6 d以上。转染24 h后的细胞流式分析显示转染的细胞中有6.0%发出荧光,说明构建的复制子载体能够有效表达EGFP蛋白。另外,间接免疫荧光、RT-PCR和Western blot方法也检测到该复制子RNA在BSR/T7细胞中能够进行自主复制,并且能够表达病毒的非结构蛋白。【结论】含有EGFP报告基因的FMDV亚基因组复制子的成功构建为进一步研究病毒复制、翻译机制及筛选抗病毒药物等奠定了坚实的基础。     

昆津病毒复制子——一种新型病毒载体

病毒复制子 (Replicon) 是指来源于病毒基因组的能够自主复制的RNA分子,保留了病毒非结构蛋白基因,而结构蛋白基因缺失或由外源基因替代。昆津病毒 (Kunjun virus) 为黄病毒科黄病毒属成员,其复制子具有表达效率高、细胞毒性低、遗传稳定等特点,在病毒基因组复制调控机制、外源蛋白表达、新型疫苗和基因治疗等领域得到了广泛应用。以下就昆津病毒复制子系统的构建、特性及应用方面的研究进展作一综述。     

发热伴血小板减少综合征布尼亚病毒微复制子的建立

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国新发现的一种布尼亚病毒,可引起人类严重发热伴血小板减少综合征。我们利用RNA聚合酶Ⅰ体系,分别构建SFTSV三个片段L、M、S微复制子,研究其非编码区调控功能。将报告基因绿色荧光蛋白(GFP)或荧光素酶(Luciferase)分别插入SFTSV三个片段5′和3′非编码区之间,所形成的嵌合cDNA反向插入含RNA聚合酶I的表达载体pHH21中,获得SFTSV微复制子重组质粒L-GFP-pHH21、M-GFP-pHH21、S-GFP-pHH21、L-Luc-pHH21、M-Luc-pHH21和S-Luc-pHH21,分别与成功表达SFTSV聚合酶蛋白(L蛋白)和结构蛋白(N蛋白)的质粒VR1012-L和VR-1012-NP共同转染293T细胞,24～48h后观察GFP表达情况或检测萤光素酶表达量。L、M、S片段GFP微复制子均可观察到特异性绿色荧光。荧光素酶定量结果显示其在不同节段非编码区中的表达量不同,提示SFTSV三个节段的非编码区启动微复制子转录和复制的强度不同。     

一种基于塞姆利基森林病毒复制子的新型复制子载体

RNA复制子是能自主复制的病毒RNA。基于RNA复制子的表达载体和基因疫苗比常规真核表达载体和DNA疫苗具有更大的优越性。以塞姆利基森林病毒RNA复制子衍生的真核表达载体pSFV1为骨架 ,插入CMV立即早期启动子和SV40晚期Poly(A)信号 ,构建了一种完全基于DNA的复制型表达载体pSFV1CS ,将增强型绿色荧光蛋白基因EGFP插入其中 ,构建了重组质粒pSFV1CS EGFP ,通过转染 2 93T细胞 ,证实外源基因能在其中高效表达。该载体可用于表达真核蛋白和构建复制子载体疫苗。     

基于寨卡病毒复制子的抗病毒药物筛选系统

寨卡病毒(Zika virus, ZIKV)是引起成人格林巴利综合征(Guillain-Barre syndrome, GBS)以及胎儿和新生儿小头畸形疾病的重要病原体。有效的抗病毒药物筛选系统是防治ZIKV感染的有效工具。本研究构建了以海肾荧光素酶为检测靶标的寨卡病毒复制子Rluc-ZIKV-Rep,并通过突变NS5蛋白,构建了非复制型寨卡病毒复制子Rluc-ZIKV-Rep NS5△GDD。为了检测复制子的功能,将两种复制子转化至Vero细胞,结果显示随着时间延长, Rluc-ZIKV-Rep荧光素酶活性逐渐增加,寨卡病毒m RNA拷贝数逐渐增多,证明以海肾荧光素酶为检测靶标的寨卡病毒复制子构建成功。为了检测该系统是否能够用于抗病毒药物筛选,用马尼地平和西尼地平两种抗寨卡病毒的药物进行检测,结果显示Rluc-ZIKV-Rep的复制能力随着药物浓度的增加而逐渐降低,呈现一种剂量依赖的方式,说明该系统可以用于抗病毒药物的筛选。综上所述,本研究构建了一种可以用于抗病毒药物筛选的寨卡病毒复制子,为筛选有效的抗病毒药物提供了有力工具。     

乙型脑炎病毒表达载体的研究

用RT-PCR方法分段扩增了乙型脑炎病毒SA14—14—2疫苗株5′、3′NCR,利用融合PCR技术在5′、3′NCR之间引入BamHⅠ酶切位点,将5′NCR置于T7启动子控制之下,构建乙脑病毒微复制子表达载体pMR。分别将绿色荧光蛋白(GFP)和汉滩病毒核蛋门编码区基因插入到pMR中,构建两种表达外源基因的乙脑病毒微复制子表达载体：pMR—GFP和pMR-84FliS。绎荧光显微镜直接观察、Western blot、ELISA等方法检测,证实外源基因能够在辅助病毒SA14—14—2感染的BHK-21细胞中表达。     

日本脑炎病毒C蛋白对其复制子载体自主复制活性的影响

为了研制具有高效自主复制能力的日本脑炎病毒 (JEV) 复制子载体,验证其作为新型复制子疫苗载体的可能性。以保留全长核心蛋白C基因的JEV复制子载体pCTCJEV为基础,通过PCR的方法减短C蛋白的部分基因序列,分别保留C23和C68位氨基酸,以Lac Z基因作为报告基因,构建了C基因长短不同的JEV复制子载体pCMW-2M-1LACZ、pCMW-2M-3LACZ。将复制子载体转染表达JEV结构蛋白的细胞系CME-4,通过Lac Z的表达检测JEV复制子载体表达外源蛋白的能力,反映了JEV的系列复制子载体的自主复制能力。结果保留C基因全长,C68、C23的复制子载体表达外源蛋白的能力相当,以上结果说明仅仅保留C蛋白的69个核苷酸即可保留JEV复制子载体的自主复制能力,为进一步优化JEV复制子载体,将该载体开发研制成为高效表达外源蛋白的疫苗载体提供了依据。     

RNA复制子疫苗及其包装系统

RNA复制子疫苗是一种基于RNA的复制子,能够进行自我复制的新型疫苗,保留了病毒的复制酶基因,结构基因由外源基因所代替,复制酶可控制载体RNA在胞质中高水平复制和外源基因高水平的表达。RNA复制子疫苗被包装成病毒样颗粒后,大大提高了RNA复制子的稳定性。近几年来,关于RNA复制子的包装系统发展迅速,并且许多包装系统都获得成功,大大提高了复制子疫苗的生物安全性和外源基因的高效表达性,具有很好的应用前景。     

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp,nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.     

Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated replicon RNA from virus stocks after 21 serial passages of the replicon genomes with VV-P1 indicated that the replicon which contained the VP4 coding region was present at a higher level than the replicon which contained a complete substitution of the P1 capsid sequences. These differences in encapsidation, though, were not detected after only two serial passages of the replicons with VV-P1 or upon coinfection and serial passage with type 1 Sabin poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV)

Rabbit hemorrhagic disease virus (RHDV), the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg) is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E) in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.     

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.     

N端融合外源蛋白的兔出血症病毒衣壳蛋白自聚能力的研究

将兔出血症病毒衣壳蛋白VP6 0基因插入杆状病毒转移载体pBLUEBACHIS2_B的 6 HIS表达标签下游 ,与线性化野生型杆状病毒基因组DNA共转染Sf9昆虫细胞 ,经蚀斑纯化后获克隆化重组杆状病毒pBLUEBACHIS2B_VP6 0。以重组杆状病毒感染Sf9细胞 ,经SDS_PAGE和Westernblot检测显示高效表达一分子量为 6 9kD的重组蛋白 ,并且该蛋白可被兔抗RHDV高免血清识别。血凝试验表明 ,该重组蛋白可以凝集人“O”型红细胞 ,血凝价达 2 1 6 ,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组病毒表达的融合有 6 HIS表达标签的衣壳蛋白仍可在昆虫细胞内自聚成不包裹核酸的、与天然RHDV病毒粒子在物理形态上相似的病毒样颗粒 (VLPs) ,并且该VLPs与兔抗RHDV高免血清作用后于电镜下可见凝集成团的现象 ,表明其与天然RHDV病毒粒子在抗原性上也极为相似     

表达兔出血症病毒VP60蛋白的重组兔粘液瘤病毒的构建与初步评价

兔出血症病毒 (Rabbit hemorrhagic disease virus,RHDV) 及兔粘液瘤病毒 (Myxoma virus,MYXV) 分别引起兔出血症 (兔瘟) 和兔粘液瘤病,是两种严重危害家兔养殖业以及导致原产地欧洲野兔-穴兔 (Oryctolagus cuniculus) 种群近濒危的重要病原。VP60为构成RHDV衣壳的主要抗原蛋白。为研制能同时免疫预防该两种疫病的重组二联疫苗,本研究分别以MYXV和其复制非必需基因——胸腺激酶 (Thymidine kinase,TK) 基因为重组载体和同源重组靶基因,构建穿梭载体p7.5-VP60-GFP。将p7.5-VP60-GFP载体转染被MYXV感染的兔肾细胞株RK13,经同源重组后,在荧光显微镜下筛选出表达GFP的重组病毒,并将其命名为rMV-VP60-GFP。通过PCR和Western blotting进行重组病毒vp60基因特异性插入和表达验证结果显示,vp60和gfp基因成功插入MYXV基因组中并且可成功表达,表明成功构建了表达RHDV衣壳蛋白基因vp60的重组MYXV。动物攻毒保护试验表明,制备的重组病毒能保护家兔抵抗MYXV的致死性攻击,这为后续疫苗的研发奠定了基础。