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.

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.

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.

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 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.

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.

Eukaryotic Expression and Activity Analysis of Capsid Gene of Swine Vesicular Disease Virus HK/70

The capsid protein precursor （P1）, which plays a major role for the generation of polypeptides of swine vesicular disease virus （SVDV）, was cloned from SVDV HK/70 strain into the retroviral vector pBABE puro and expressed in the mammalian cell line PK15 through the retroviral expression system. The activity of recombinant protein to induce immune response was evaluated in guinea pigs. IFA and Western Blot were used to detect the recombinant protein expression. The results showed that the recombinant protein could be recognized by SVDV positive serum, and animal test showed SVDV-specific antibodies. All of those results indicate that a retroviral-based vaccine carrying the capsid protein precursor （P1） of SVD is able to be expressed in the eukaryotic cell and elicites strong SVDV-specific immune responses in guinea pigs.

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.

Functional Inferences of Environmental Coccolithovirus Biodiversity

The cosmopolitan calcifying alga Emiliania huxleyi is one of the most abundant bloom forming coccolithophore species in the oceans and plays an important role in global biogeochemical cycling. Coccolithoviruses are a major cause of coccolithophore bloom termination and have been studied in laboratory, mesocosm and open ocean studies. However, little is known about the dynamic interactions between the host and its viruses, and less is known about the natural diversity and role of functionally important genes within natural coccolithovirus communities. Here, we investigate the temporal and spatial distribution of coccolithoviruses by the use of molecular fingerprinting techniques PCR, DGGE and genomie sequencing. The natural biodiversity of the virus genes encoding the major capsid protein （MCP） and serine palmitoyltransferase （SPT） were analysed in samples obtained from the Atlantic Meridional Transect （AMT）, the North Sea and the L4 site in the Westem Channel Observatory. We discovered nine new coccolithovirus genotypes across the AMT and L4 site, with the majority of MCP sequences observed at the deep chlorophyll maximum layer of the sampled sites on the transect. We also found four new SPT gene variations in the North Sea and at L4. Their translated fragments and the full protein sequence of SPT from laboratory strains EhV-86 and EhV-99B 1 were modelled and revealed that the theoretical fold differs among strains. Variation identified in the structural distance between the two domains of the SPT protein may have an impact on the catalytic capabilities of its active site. In summary, the combined use of ＇standard＇ markers （i.e. MCP）, in combination with metabolically relevant markers （i.e. SPT） are useful in the study of the phylogeny and functional biodiversity of coccolithoviruses, and can provide an interesting intracellular insight into the evolution of these viruses and their ability to infect and replicate within their algal hosts.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

Establishment of Real-time Fluorescent RT-LAMP Detection Method for Grouper Nervous Necrosis Virus

Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of red-spotted grouper nervous necrosis virus （NNV）. By optimizing the reaction conditions, a rapid and simple reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was established for NNV detection. After adding SYTO-9 fluorescent dye in the reaction system, the amplification curve was monitored in real time using a fluorescence detector, and the result was obviously easy to assess. Moreover, the specificity and sensitivity of the established method was analyzed. The results showed that the established RT-LAMP method has good specificity with a detection limit of 1.3 pg/μl. The detection sensitivity of the established RT-LAMP method is 100 times that of the conventional RT-PCR method, and the detection duration is only 40 min. The established RT-LAMP method is suitable for quarantine and rapid detection of grouper nervous necrosis virus.

Generation and Characterization of Monoclonal Antibodies Against Red-Spotted Grouper Nervous Necrosis Virus(RGNNV)

Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.

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.

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.

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.

Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Cytomegalovirus(CMV)is distinct among members of the Herpesviridae family for having the largest dsDNA genome(230 kb).Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid,but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described.Here,we report a cryo electron microscopy(cryoEM)structure of the murine cytomegalovirus(MCMV)capsid at a 9.1Åresolution and describe the molecular interactions among the~3000 protein molecules in the MCMV capsid at the secondary structure level.Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling.The major capsid protein(MCP)upper domain(MCPud)containsα-helices andβ-sheets conserved with those in MCPud of herpes simplex virus type 1(HSV-1),with the largest differences identifi ed as a“saddle loop”region,located at the tip of MCPud and involved in interaction with the smallest capsid protein(SCP).Interactions among the bacteriophage HK97-like fl oor domain of MCP,the middle domain of MCP,the hook and clamp domains of the triplex proteins(hoop and clamp domains of TRI-1 and clamp domain of TRI-2)contribute to the formation of a mature capsid.These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attach-ing unique functional tegument proteins outside.