Bioinformatic Analysis of Non-VP1 Capsid Protein of Coxsackievirus A6

This study bioinformatically analyzed the non-VP1 capsid proteins（VP2-VP4） of Coxasckievirus A6（CVA6）, with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools Sub Loc, Target P and the others from Ex PASy Bioinformatics Resource Portal, and SWISS-MODEL（an online protein structure modeling server）, were utilized to analyze the amino acid（AA） sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices（AI） of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.

Prokaryotic Expression and Potential Application of the Truncated PCV-2 Capsid Protein

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a（＋） vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta（DE3） E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % （73/264）. Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-20RF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Expression and Immunological Analysis of Capsid Protein Precursor of Swine Vesicular Disease Virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichia pastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein(HPV16 L1) in Pichia pastoris

In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.

A Novel Pharmacophore Model Derived from a Class of Capsid Protein Enterovirus 71 Inhibitors

Capsid protein enterovirus 71 （EV71） is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis （Hypo1） has a high predictive power and consists of four features,namely,one hydrophobic point （HY） and three hydrogen-bond acceptors （HA）.Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.

Research Progress of HPV L1 Capsid Protein in Prediction of Cervical Lesions

Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females.Chronic and persistent infection with High Risk Human Papillomavirus(HR-HPV)is the main cause of cervical cancer.There is a distinct lack of methodology by which to determine whether cervical epithelial dysplasia is cancerous following HPV infection.HPV L1 capsid protein is a major structural protein of human papillomavirus(HPV),and it is the main target of the local cellular immune response aiming to combat human papillomavirus after HPV infection within cervical cells.Greater understanding of HPV L1 capsid protein and its association with cervical cytology,histopathology,patient age and human papillomavirus viral load has the potential to contribute toward improved the diagnosis and management of cervical cancer,providing useful information for gynecological clinicians in the hope of improving patient treatment and quality of life.This article reviews the predictive utility of HPV L1 capsid protein for cervical lesions.

Development of an indirect immunofluorescence assay for PCV3 antibody detection based on capsid protein

Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.

Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis

Porcine circovirus type 2（PCV2）has recently been reported to elicit the unfolded protein response（UPR）via activation of the PERK/e IF2α（RNA-activated protein kinase-like endoplasmic reticulum（ER）kinase/eukaryotic initiation factor 2α）pathway.This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein.By transient expression,we found that both replicase（Rep）and capsid（Cap）proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eI F2α-ATF4（activating transcription factor 4）-CHOP（CCAAT/enhancer-binding protein homologous protein）axis.Cap expression,but not Rep,significantly reduced antiapoptotic B-cell lymphoma-2（Bcl-2）and increased caspase-3 cleavage,possibly due to increased expression of CHOP.Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression,caspase-3cleavage,and apoptotic cell death possibly by partially rescuing Bcl-2 expression,we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eI F2α/ATF4/CHOP/Bcl-2 pathway.This study,together with our earlier studies,provides insight into the mechanisms underlying PCV2 pathogenesis.

Dissection the endocytic routes of viral capsid proteins-coated upconversion nanoparticles by single-particle tracking

Real-time exploring the cellular endocytic pathway of viral capsid proteins(VCPs)functionalized nanocargos at the single-particle level can provide deep insight into the kinetic information involved in virus infection.In this work,porcine circovirus type 2(PCV2)VCPs with different functions are modified onto the surface of upconversion nanoparticles(VCPs-UCNPs)to investigate the cellular internalization process in real-time.Clathrin-mediated endocytosis is found to be the essential uptake mechanism for these VCPs-UCNPs.Besides,it is verified that P_(1)-UCNPs(PCV2 VCPs with nuclear localization signal,namely P1)can be easily assembled close to the perinuclear area,which is different from that of P_(2)-UCNPs(PCV2 VCPs without nuclear localization signal,namely P_(2)).Interestingly,multistep entry processes are observed.Particularly,confined diffusion is observed during the transmembrane process.The intracellular transport of VCPs-UCNPs is dependent on microtubules toward the cell interior.During this process,P_(1)-UCNPs display increased velocities with active transport,while diffusion much faster around the perinuclear area.But for P_(2)-UCNPs,there are only two phases involved in their endocytosis process.This study presents distinct dynamic mechanisms for the nanocargos with different functions,which would make a useful contribution to the development of robust drug delivery systems.

Novel PF74-like small molecules targeting the HIV-1 capsid protein:Balance of potency and metabolic stability

Of all known small molecules targeting human immunodeficiency virus(HIV)capsid protein(CA),PF74 represents by far the best characterized chemotype,due to its ability to confer antiviral phenotypes in both early and late phases of viral replication.However,the prohibitively low metabolic stability renders PF74 a poor antiviral lead.We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site.Specifically,we replaced the inter-domain-interacting,electron-rich indole ring of PF74 with less electron-rich isosteres,including imidazolidine-2,4-dione,pyrimidine-2,4-dione,and benzamide,and identified four potent antiviral compounds(10,19,20 and 26)with markedly improved metabolic stability.Compared to PF74,analog 20 exhibited similar submicromolar potency,and much longer(51-fold)half-life in human liver microsomes(HLMs).Molecular docking corroborated that 20 binds to the PF74 binding site,and revealed distinct binding interactions conferred by the benzamide moiety.Collectively,our data support compound 20 as a promising antiviral lead.

3D reconstruction and capsid protein characterization of grass carp reovirus

Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic se-quencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 ? resolution by electron cryomicro-scopy. Each GCRV capsid has a multilayered organization, consisting of an RNA core, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression re-sulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symme-try. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin

Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备

【目的】本研究选择原核表达系统表达鸭坦布苏病毒(Duck Tembusu virus,DTMUV)核衣壳蛋白(Capsid protein),并制备其多克隆抗体,为DTMUV分子机制研究奠定基础。【方法】根据DTMUV-201909株基因序列,运用一步克隆技术将Capsid基因克隆至表达载体pET-30a(+)中,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG进行诱导表达,通过SDS-PAGE和Western blotting鉴定重组蛋白;使用ISA206佐剂与纯化后的重组蛋白混合乳化后免疫BALB/c小鼠,以获得多克隆抗体。间接ELISA方法测定获得的多克隆抗体效价,并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)验证。【结果】试验成功构建pET-30a-Capsid重组质粒,SDS-PAGE结果显示,表达的重组蛋白大小约为18 ku,主要以包涵体形式存在;Western blotting结果表明,该蛋白能与抗His标签鼠单克隆抗体发生特异性反应,具有良好反应原性。间接ELISA结果显示,制备的鼠抗Capisd蛋白多克隆抗体效价可达1∶256000;Western blotting和IFA结果显示,制备的多克隆抗体能特异性识别DTMUV感染细胞样品的Capsid蛋白。【结论】本研究成功制备小鼠抗Capsid蛋白多克隆抗体,为深入研究DTMUV Capsid蛋白的结构和功能提供了试验材料,为进一步阐明DTMUV的致病机制奠定基础。

Homology Modelling and Structural Comparisons of Capsid-Associated Proteins from Circoviruses Reveal Important Virus-Specific Surface Antigens

Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their hosts. The capsid-associated protein (Cap) of circoviruses is of interest because of its role in viral structure, immune evasion, host cell entry, and nuclear shuttling of viral components. The structure of the porcine circovirus 2 (PCV2) Cap has been solved and offered insight to these functions. Based on the crystallographic PCV2 Cap structure, models from circoviruses isolated from avian, fish, and mammalian hosts have been constructed and analyzed to better understand the roles of these proteins in the virus family. A high degree of conservation is observed in the models, however, the surface antigens differ among viruses. This is likely a reflection of the small genome harbored by circoviruses, and therefore the requirement of their few proteins to carry out specific vital functions, while maintaining enough variation to successfully infect their hosts. Here we describe the putative structures of a range of Cap proteins from circoviruses based on the crystallographic determination of porcine Cap, identifying key regions for function and inhibition of crystal formation.

Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.

Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

大口黑鲈蛙虹彩病毒MCP蛋白多克隆抗体的制备与应用

【目的】制备大口黑鲈蛙虹彩病毒主衣壳(MCP)蛋白兔多克隆抗体,以期为该病毒蛋白功能研究奠定基础。【方法】对pET32a(+)MCP/BL21重组大肠杆菌进行诱导表达,将重组蛋白进行纯化、复性,以此为抗原免疫大耳兔制备多克隆抗体,ELISA检测抗体效价,间接免疫荧光试验(IFA)和Western Blot法分析MCP多克隆抗体的特异性。【结果】纯化的MCP重组蛋白条带特异;间接ELISA结果显示,制备的兔多克隆抗体血清效价为1∶1024000;IFA和Western Blot检测结果表明该多抗特异性良好,能够与大口黑鲈蛙虹彩病毒MCP蛋白发生特异性反应,IFA试验表明血清最适稀释度为1∶500。【结论】成功制备了大口黑鲈蛙虹彩病毒MCP兔多克隆抗体,该抗体可特异性识别大口黑鲈蛙虹彩病毒MCP蛋白。

嵌杯病毒主要衣壳蛋白的研究进展

嵌杯病毒(calicivirus)是无囊膜的单股正链RNA病毒,具有广泛的宿主范围因而严重威胁着人和许多其他脊椎动物的健康。由于该病毒种属流行株多变、极易传播且呈现区域性感染,致使全球范围内因杯状病毒引起的公共卫生疾病频繁暴发,近年来,其感染发病率呈明显增加的趋势。不同属成员虽然感染不同种类的宿主,但其主要衣壳蛋白的结构类似。嵌杯病毒主要衣壳蛋白中含有免疫原性关键区域,能与宿主受体结合并可能介导感染过程,且氨基酸相关的免疫原性及受体结合位点的变异是公认的病毒进化的驱动力。本文就嵌杯病毒主要衣壳蛋白的基因组结构、免疫原性、受体结合情况以及遗传演化规律等方面的研究进展进行综述,以期为后续科学研究以及该病的有效防控提供参考。