Capsid Protein VP1 Research Articles

Identification of a blockade epitope of human norovirus GII.17

ABSTRACT Human noroviruses are the dominant causative agent of acute viral gastroenteritis worldwide. During the winter of 2014–2015, genotype GII.17 cluster IIIb strains emerged as the leading cause of norovirus infection in Asia and later spread to other parts of the world. It is speculated that mutation at blockade epitopes may have resulted in virus escape from herd immunity, leading to the emergence of GII.17 cluster IIIb variants. Here, we identify a GII.17 cluster IIIb-specific blockade epitope by monoclonal antibody (mAb)-based epitope mapping. Four mAbs (designated as M1 to M4) were generated from mice immunized with virus-like particle (VLP) of a GII.17 cluster IIIb strain. Among them, M1 and M3 reacted specifically with the cluster IIIb VLP but not with the VLPs from clusters II or IIIa. Moreover, M1 and M3 dose-dependently blocked cluster IIIb VLP binding with its ligand, histo-blood group antigens (HBGAs). Epitope mapping revealed that M1 and M3 recognized the same highly exposed epitope consisting of residues 293–296 and 299 in the capsid protein VP1. Sequence alignment showed that the M1/M3 epitope sequence is highly variable among different GII.17 clusters whereas it is identical for cluster IIIIb strains. These data define a dominant blockade epitope of GII.17 norovirus and provide evidence that blockade epitope evolution contributes to the emergence of GII.17 cluster IIIb strains.

ИСПОЛЬЗОВАНИЕ РЕКОМБИНАНТНОГО БЕЛКА VP2 В КАЧЕСТВЕ СУБЪЕДИНИЧНОЙ ВАКЦИНЫ ПРОТИВ ИНФЕКЦИОННОЙ БУРСАЛЬНОЙ БОЛЕЗНИ

In all poultry producing regions of the world, infectious bursal disease (IBD) virus continues to be a major concern for poultry farmers. The virus causes anemic and dehydrated muscle tissue in chickens, as well as hemorrhages in the muscles of the lower leg, thigh, wings and chest. The most characteristic of this disease are observed in the fabrication bursa. In the first 2-4 days after infection of the bird, it increases 2-3 times. Its mucous membrane becomes edematous, hyperemic, hemorrhages and necrotic areas are found in it. The mortality from this disease is low, but the main danger of IBD is the immunosuppressive state it causes. The consequences of immunosuppression associated with IBD, an increase in the susceptibility of chickens to opportunistic microorganisms, an increase in susceptibility to diseases and a decrease in the effectiveness of vaccination against Newcastle disease, Marek's disease, infectious bronchitis, etc. Vaccination is the most important measure for combating IBD in the field. Today, live and inactivated vaccines against infectious bursal disease are widely used in poultry farms. A large number of disadvantages of their use have been found, therefore, the relevance of recombinant vaccines based on the viral VP2 protein has increased, which are considered safer and cheaper. Vaccinated chickens, in which the synthesis of virus-neutralizing antibodies to the VP2 capsid protein occurs, are resistant to infectious bursal disease. The aim of the work was the recombinant protein VP2 and to test its effectiveness in chickens of the Lohman Brown cross.

Tropism of Coxsackie virus A9 depends on the +1 position of the RGD (arginine- glycine- aspartic acid) motif found at the C’ terminus of its VP1 capsid protein

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvβ6 and/or αvβ3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvβ3 and αvβ6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy.

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge

Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective after a BTV-4 boost. In sheep, a natural host for RVFV and BTV, both vaccines proved immunogenic although conferred only partial protection after a virulent BTV-4 reassortant Morocco strain challenge. Though additional optimization will be needed to improve the efficacy data against BTV in sheep, our findings warrant further developments of attenuated RVFV as a dual vaccine platform carrying heterologous immune relevant antigens for ruminant diseases in RVF risk areas.

Contemporary circulating enterovirus D68 strains have acquired the capacity for viral entry and replication in human neuronal cells

Background: Enterovirus D68 (EV-D68) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. EV-D68 infection has historically been associated with respiratory illnesses in humans. However, in the summers of 2014, 2016, and 2018 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis (AFM) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. Methods and materials: To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a cell culture model to determine if differential infection is observed for different EV-D68 isolates. Viral replication and infectivity in SH-SY5Y was assessed using four different assays-infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Results: In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to virus binding and entry were at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. Conclusion: Taken together, these results suggest that EV-D68 has evolved to acquire novel neurotropic characteristics that may contribute to the development of neurological disease in humans. In addition to supporting virus replication and other functional studies, the cell culture model we’ve developed may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Circulation of diverse protoparvoviruses in wild carnivores, Italy.

Protoparvovirus is a monophyletic viral genus that includes the species Carnivore protoparvovirus-1 infecting domestic and wild carnivores. In this paper, the results of an epidemiological survey for Carnivore protoparvovirus-1 in wild carnivores in Italy are reported. Overall, 34 (11.4%) out of 297 tested animals were positive for Carnivore protoparvovirus-1, but the frequency of detection was much higher in intestine (54%) than in spleen samples (2.8%), thus suggesting that the intestine is the best sample to collect from wild animals for parvovirus detection. Feline panleukopenia virus (FPV) was detected in red foxes (Vulpes vulpes) (2.8%, 7/252) and Eurasian badgers (Meles meles) (10%, 1/10), whilst canine parvovirus (CPV) was found in wolves (54.3%, 19/35), Eurasian badgers (60%, 6/10) and one beech marten (Martes foina) (100%, 1/1), with more than one parvovirus type detected in some animals. Protoparvoviral DNA sequences from this study were found to be related to CPV/FPV strains detected in Asia and Europe, displaying some amino acid changes in the main capsid protein VP2 in comparison with other parvovirus strains from wildlife. In particular, the two most common mutations were Ile418Thr and Ala371Gly, which were observed in 6/12 (50%) and 5/12 (41.7%) of the CPV sequences from this study. Continuous surveillance for parvoviruses in wild carnivores and genetic analysis of the detected strains may help obtain new insight into the role of these animals in the evolution and epidemiology of carnivore parvoviruses.

Self-Assembled Multi-Epitope Peptide Amphiphiles Enhance the Immune Response against Enterovirus 71.

Subunit vaccines consist of non-genetic material, such as peptides or proteins. They are considered safe because they have fewer side effects; however, they have low immunogenicity when used alone. We aimed to enhance the immune response of peptide-based vaccines by using self-assembled multimeric peptide amphiphiles (PAs). We designed two epitope PAs by conjugating epitope peptides from Enterovirus 71 (EV71) virus particle (VP) 1 and VP3 capsid proteins with different fatty acid chain lengths (VP1PA and VP3PA). These PAs self-assembled into supramolecular structures at a physiological pH, and the resulting structures were characterized using atomic force microscopy. Multi-epitope PAs (m-PAs) consisted of a 1:1 mixture of VP1PA and VP3PA solutions. To evaluate immunogenicity, m-PA constructs were injected with adjuvant subcutaneously into female Balb/c mice. Levels of antigen-specific immunoglobulin G (IgG) and IgG1 in m-PA-injected mice serum samples were analyzed using ELISA and Western blotting. Additionally, cytokine production stimulated by each antigen was measured in splenocytes cultured from immunized mice groups. We found that m-PA showed improved humoral and cellular immune responses compared to the control and peptide groups. The sera from m-PA immunized mice group could neutralize EV71 infection and protect host cells. Thus, self-assembled m-PAs can promote a protective immune response and can be developed as a potential platform technology to produce peptide vaccines against infectious viral diseases.

Antigenicity study of the yeast-generated human parvovirus 4 (PARV4) virus-like particles

Human parvovirus 4 (PARV4) is a novel tetraparvovirus that was isolated from intravenous drug users in 2005. Recombinant PARV4 capsid protein VP2 can form stable virus-like particles (VLPs) in yeast. These VLPs could act as antigen carriers during vaccine development. Therefore, the information about PARV4 VP2 VLP antigenic sites could advance further research in this area. In this work, human parvovirus 4 VLPs obtained from yeast were used to generate monoclonal antibodies (mAbs) in mice. Epitope mapping of the obtained mAbs showed at least three distinct antigenic sites of the VP2 protein. On top of that, molecular cloning was used to replace PARV4 VP2 antigenic sites with heterologous peptides. The chimeric PARV4 VLPs bearing polyhistidine inserts obtained from yeast were observed using electron microscopy while polyhistidine-specific antibodies detected heterologous peptides of the chimeric VP2 proteins.

Comparison of Immunohistochemical Staining for Large T Antigen and Capsid Protein VP1 in BK Polyomavirus-Associated Nephropathy

Aim: Most transplant centres use SV40 large T antigen (TAg) staining for the diagnosis and assessment of BK polyomavirus-associated nephropathy (BKPyVAN). This study was performed to evaluate the significance of capsid protein VP1 expression in BKPyVAN. Methods: We performed immunohistochemical staining using anti-SV40 TAg and anti-BKPyV VP1 antibodies in 16 index biopsies and 12 re-biopsies of BKPyVAN and compared the patterns of positivity and the percentage of positive tubules by counting whole specimens. We investigated the correlation between serum creatinine increase from baseline and the percentage of positive tubules for both markers in 16 index biopsies. Results: In VP1 staining, positive findings were observed not only in the nuclei of tubular epithelial cells but also in the cytoplasm, cells shedding into the lumen, intra-tubular casts, and in the interstitium. Two of 28 biopsies (7.1%) showed TAg-positive and VP1-negative results, in which TAg-positive cells were detected only in a single tubule. The median (interquartile range) percentage of positive tubules was 2.8% (0.7–9.8%) for TAg and 1.4% (0.5–3.9%) for VP1 staining (p = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (r = 0.49, p = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. Conclusions: VP1 expression showed various patterns, but was detected in half as many tubules as TAg staining, which might lead to false negatives in the samples with minimal viral replication. However, increased VP1-positive tubules indicate advanced tubular damage and possible association with graft dysfunction.

Detection of Parechovirus A1 with Monoclonal Antibody against Capsid Protein VP0.

Parechovirus A (PeV-A; human parechovirus) causes mild infections and severe diseases such as neonatal sepsis, encephalitis, and cardiomyopathy in young children. Among the 19 types of PeV-A, PeV-A1 is the most common type of infection. We have previously established an immunofluorescence assay for detecting multiple PeV-A types with a polyclonal antibody against the conserved epitope of VP0. Although the polyclonal antibody is useful for PeV-A diagnosis, it could not distinguish the PeV-A genotypes. Thus, the development of a specific monoclonal antibody for identifying the common infection of PeV-A1 would be beneficial in clinical diagnosis practice. In this study, the recombinant full-length PeV-A1 VP0 protein was used in mouse immunization; a total 10 hybridomas were established. After evaluation by immunoblotting and fluorescence assays, six hybridoma clones with monoclonal antibody (mAb) production were confirmed. These mAbs, which specifically recognize viral protein PeV-A1 VP0 without cross-reactivity to PeV-A3, will prove useful in research and PeV-A1 diagnosis.

Hsp90 is involved in pseudorabies virus virion assembly via stabilizing major capsid protein VP5

Many viruses utilize molecular chaperone heat shock protein 90 (Hsp90) for protein folding and stabilization, however, the role of Hsp90 in herpesvirus lifecycle is obscure. Here, we provide evidence that Hsp90 participates in pseudorabies virus (PRV) replication. Viral growth kinetics assays show that Hsp90 inhibitor geldanamycin (GA) abrogates PRV replication at the post-penetration step. Transmission electron microscopy demonstrates that dysfunction of Hsp90 diminishes the quantity of PRV nucleocapsids. Overexpression and knockdown of Hsp90 suggest that de novo Hsp90 is involved in PRV replication. Mechanismly, dysfunction of Hsp90 inhibits PRV major capsid protein VP5 expression. Co-immunoprecipitation and indirect immunofluorescence assays indicate that Hsp90 interacts with VP5. Interestingly, Hsp70, a collaborator of Hsp90, also interacts with VP5, but doesn't affect PRV growth. Finally, inhibition of Hsp90 results in PRV VP5 degradation in a proteasome-dependent manner. Collectively, our data suggest that Hsp90 contributes to PRV virion assembly and replication via stabilization of VP5.

Non-muscle myosin heavy chain 9 is a critical factor for infectious pancreatic necrosis virus cellular entry

Infectious pancreatic necrosis virus (IPNV) is a highly pathogenic agent of salmonid. However, the critical factors responsible for IPNV binding largely remain unknown. Here, by using a combination of immunoprecipitation and mass spectrometry assays, we identified that non-muscle myosin heavy chain 9 (Myh9) played an important role in IPNV entry by interacting with viral outer capsid protein VP2 on cell surface. We also discovered that IPNV infection induced redistribution of Myh9 to the cell membrane at the initiation of viral entry and altered the expression level of total Myh9 during viral replication. Treatments with different Myh9 inhibitors, including anti-Myh9 antibody, ML-7 and siRNA, suppressed IPNV infection. On the contrary, overexpression of Myh9 promoted IPNV infection in both susceptible and less susceptible cells. By using quantum dots-labeling technology and confocal microscopy, we tracked the entry of IPNV in real-time and found the adsorbed virions increased significantly in Myh9 overexpressed cells. Additionally, we found IPNV infection induced an increase of intracellular nanotubes and the virus could move along nanotubes to neighboring cells. Overall, our results indicate that Myh9 plays a substantial role in the entry of IPNV.

Effect of a Neuropilin-1-Derived Virus Receptor Trap on Enterovirus A71 Infection In Vitro.

We discovered that neuropilin 1 (NRP1) is a new receptor candidate to mediate enterovirus A71 (EVA71) into cells. In the engineered form as a decoy receptor, NRP1 was able to recognize and neutralize EVA71 but not enterovirus D68 or coxsackievirus B3 (CVB3). NRP1 recognizes EVA71 through a novel domain on the VP3 capsid protein. The principle in the design, engineering, and refinement of the NRP1-based decoy receptor described in this study represents a general and well-suited antiviral strategy.

Obtaining recombinant capsid protein VP60 of rabbit haemorrhagic disease virus and its antigenic and immunogenic activity study

Viral hemorrhagic disease of rabbits is an acute, highly contagious disease characterized by the phenomena of hemorrhagic diathesis in all organs, especially in the lungs and liver. The causative agent of viral haemorrhagic disease of rabbits is a virus of haemorrhagic disease of rabbits, belonging to the family Caliciviridae, genus Lagovirus. Currently, there are 4 genogroups of lagoviruses, two pathogenic: GI1 (GI1a-GI1d) and GI2, and two non-pathogenic: GI3 and GI4. The greatest danger to rabbits in the Russian Federation is posed by viruses of the GI1 genotype. The virulence of these viruses for rabbits is extremely high, the incubation period is 48-72 hours. Clinically, the disease is almost not manifested. Mortality can reach 100%. For the prevention of HBV in the Russian Federation, inactivated tissue vaccines are used, which are a suspension of the liver of rabbits infected with virulent strains of the rabbit hemorrhagic disease virus. Currently, in veterinary practice, subunit recombinant vaccines based on virus proteins obtained in the baculovirus gene expression system are increasingly used. The authors obtained the recombinant VP60 virus of rabbit hemorrhagic disease of the GI1 genotype in the baculovirus gene expression system and studied its antigenic and immunogenic activity for rabbits. It was found that the recombinant capsid protein VP60 of the hemorrhagic disease virus, administered to rabbits at a dose of 50 pg, causes the synthesis of specific antibodies in animals, detected by enzyme immunoassay and in the hemagglutination inhibition reaction, and protects 80% of animals during control infection with the virulent strain «Voronezh-87» at a dose of 1000 LD 50. These data indicate the possibility of using this protein as a specific component of a subunit vaccine against rabbit hemorrhagic disease caused by strains of the GI1 gene group.

Generation and evaluation of a recombinant myxomavirus expressing the VP60 protein of rabbit haemorrhagic disease virus

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.

SV40 Polyomavirus Activates the Ras-MAPK Signaling Pathway for Vacuolization, Cell Death, and Virus Release.

Polyomaviruses are a family of small, non-enveloped DNA viruses that can cause severe disease in immunosuppressed individuals. Studies with SV40, a well-studied model polyomavirus, have revealed the role of host proteins in polyomavirus entry and trafficking to the nucleus, in viral transcription and DNA replication, and in cell transformation. In contrast, little is known about host factors or cellular signaling pathways involved in the late steps of productive infection leading to release of progeny polyomaviruses. We previously showed that cytoplasmic vacuolization, a characteristic late cytopathic effect of SV40 infection, depends on the specific interaction between the major viral capsid protein VP1 and its cell surface ganglioside receptor GM1. Here, we show that, late during infection, SV40 activates a signaling cascade in permissive monkey CV-1 cells involving Ras, Rac1, MKK4, and JNK to stimulate SV40-specific cytoplasmic vacuolization and subsequent cell lysis and virus release. Inhibition of individual components of this signaling pathway inhibits vacuolization, lysis, and virus release, even though high-level intracellular virus replication occurs. Identification of this pathway for SV40-induced vacuolization and virus release provides new insights into the late steps of non-enveloped virus infection.

Individual subunits of a rhinovirus causing common cold exhibit largely different protein-RNA contact site conformations

Rhinoviruses cause the common cold. They are icosahedral, built from sixty copies each of the capsid proteins VP1 through VP4 arranged in a pseudo T = 3 lattice. This shell encases a ss(+) RNA genome. Three-D classification of single and oligomeric asymmetric units computationally excised from a 2.9 Å cryo-EM density map of rhinovirus A89, showed that VP4 and the N-terminal extension of VP1 adopt different conformations within the otherwise identical 3D-structures. Analysis of up to sixty classes of single subunits and of six classes of subunit dimers, trimers, and pentamers revealed different orientations of the amino acid residues at the interface with the RNA suggesting that local asymmetry is dictated by disparities of the interacting nucleotide sequences. The different conformations escape detection by 3-D structure determination of entire virions with the conformational heterogeneity being only indicated by low density. My results do not exclude that the RNA follows a conserved assembly mechanism, contacting most or all asymmetric units in a specific way. However, as suggested by the gradual loss of asymmetry with increasing oligomerization and the 3D-structure of entire virions reconstructed by using Euler angles selected in the classification of single subunits, RNA path and/or folding likely differ from virion to virion.

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3.

Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.

SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms.

Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.