Viral Capsid Protein Research Articles

IgG Seroreactivites to Viral Capsid Protein VP1 of JC and BK Polyomaviruses in Children at Early Ages with Special Reference to Parental Cofactors.

BK (BKPyV) and JC (JCPyV) polyomaviruses are widespread in humans. Transmission at an early age and the role of parents in spreading these viruses through the family are incompletely understood. Our aim was to determine the seroprevalence of BKPyV and JCPyV in infants at the age of 1, 2, 6, 12, 24, and 36 months and to assess the frequency of BKPyV and JCPyV seroconversion. A variety of maternal and paternal covariates were also tested as potential predictors of these early childhood infections. We used multiplex serology to analyze antibodies to BKPyV and JCPyV from baseline to 3-year follow-up visits. We observed that there was nearly perfect correlation in BKPyV and JCPyV serum IgG antibody levels between the mother-infant pairs during the first year of the infant's life. No correlation among BKPyV antibody titers were found in father-child pairs, whereas JCPyV antibody levels of the father and child had a significant correlation at the 2-year follow-up visit. BKPyV infection may be associated with a child's predisposition to allergy. In conclusion, after the decay of maternal antibodies, children start to develop their own immunity toward BKPyV and JCPyV, and horizontal transmission of infection in the family can occur.

Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization.

Recombinant adeno-associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale-up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity. To identify potential factors that restrained the productivity, we systematically characterized virus production kinetics through targeted quantitative proteomics and various physical assays of viral components. We demonstrated that reducing the excessive expression of gene of interest by its conditional expression greatly increased the productivity of these synthetic cell lines. Further enhancement was gained by optimizing induction profiles and alleviating proteasomal degradation of viral capsid protein by the addition of proteasome inhibitors. Altogether, these enhancements brought the productivity close to traditional multiple plasmid transfection. The rAAV produced had comparable full particle contents as those produced by conventional transient plasmid transfection. The present work exemplified the versatility of our synthetic biology-based viral vector production platform and its potential for plasmid- and virus-free rAAV manufacturing.

Porcine Astrovirus Detection and Characterization in Healthy and Diarrheic Pigs from Haryana, India

Background: Porcine astrovirus (PAstV) is an emerging pathogen detected from the feces of both healthy and diarrheic pigs with limited studies comparing the presence of PAstV and diarrhea in pigs. PAstV has been reported worldwide with a few reports from India. In addition, there are limited reports on variation in the amino acid sequence of the ORF2 region, which codes for viral capsid protein. The present study was aimed to study the presence of PAstV, association with diarrhea in pigs of various age groups and its molecular characterization from Haryana, a northern Indian state. Methods: A total of 176 rectal swabs of pigs were collected from different parts of Haryana. The samples were screened for PAstV by RT-PCR of the partial ORF1b/ORF2 genomic region. The positive samples were characterized by molecular typing and phylogenetic analysis. Result: In the present study, PAstV was detected in pigs in 16.47% of fecal samples, including 21/127 (16.53%) diarrheic and 8/49 (16.32%) non-diarrheic. The results indicated that weaning piglets were more susceptible to PAstV infection followed by suckling piglets. The phylogenetic analysis revealed the circulation of PAstV4 and PAstV2 lineages in Haryana. The study revealed endemic presence of virus in the region with high genetic diversity among the PAstV strains, suggesting a wide range of heterogeneity.

Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation

Recombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion exchange chromatography (AEX) is an attractive method for separating empty and full AAV capsids because of its scalability. Resin types and buffer composition are key considerations for AEX and must support capsid stability to be suitable for downstream processing. We examined the impact of binding durations (0-8 h) using various binding ionic strengths (15-75mM), pH (7.5-9.0), resin chemistry (POROS XQ, POROS HQ, POROS I, and BIA QA monolith), and proprietary Q resins with different ligand densities for effects on capsid stability. Empty capsids were altered upon extended binding, leading to retention time shifts and loss of resolution between empty and full capsids. Viral capsid protein analysis reveals that full capsids have more viral capsid protein 3 (VP3) proteins than empty capsids. Analytical hydrophilic liquid chromatography showed that empty capsid retention time shift is accompanied by changes to the empty capsid's native VP3 protein. Among the potential stabilizing additives considered, magnesium chloride was the most effective at reducing negative impacts caused by extended binding.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S.aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Porcine circovirus type 2 infection inhibits macrophage M1 polarization induced by other pathogens via viral capsid protein and host gC1qR protein

Porcine circovirus type 2 (PCV2) has been proven to co-infect with a variety of pathogens and cause immunosuppression. Previously, we have reported that PCV2 infection attenuates the production of pro-inflammatory cytokines induced by other pathogens in porcine macrophages. However, whether PCV2 can affect M1-type macrophage polarization induced by other pathogens is less well reported. Herein, we found that PCV2 infection suppressed M1 macrophage production induced by porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (H. parasuis) in the lung and promoted the proliferation of these pathogens in the piglets. Consistently, we confirmed that PCV2 inhibits M1 macrophage production and its associated gene expression in porcine alveolar macrophages (PAMs) both ex vivo and in vitro. Meanwhile, PCV2 inhibited lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro in a time- and dose-dependent manner. In PCV2-infected cells, LPS-induced signal transducer and activator of transcription (STAT1) phosphorylation and its nuclear translocation were decreased. Based on these findings, we further identified a role for PCV2 capsid protein (Cap) in LPS-induced M1 macrophage-associated genes and found that PCV2 Cap can significantly reduce STAT1 phosphorylation and its nuclear translocation, as well as the production of M1 macrophage-related genes. As the binding protein of PCV2 Cap, gC1qR protein was also associated with this inhibition process. gC1qR-binding activity-deficient PCV2 Cap mutated protein (Cap RmA) appeared an attenuated inhibitory effect on other pathogen-induced polarization of M1-type macrophages, suggesting that the inhibitory effect of PCV2 infection on M1-type macrophage polarization induced by other pathogens is dependent on Cap protein and the host gC1qR protein. Altogether, our results demonstrate that PCV2 infection inhibits macrophage M1 polarization induced by other pathogens via capsid and host gC1qR protein modulating JAK/STAT signaling.

Comparison, Analysis, and Molecular Dynamics Simulations of Structures of a Viral Protein Modeled Using Various Computational Tools.

The structural analysis of proteins is a major domain of biomedical research. Such analysis requires resolved three-dimensional structures of proteins. Advancements in computer technology have led to progress in biomedical research. In silico prediction and modeling approaches have facilitated the construction of protein structures, with or without structural templates. In this study, we used three neural network-based de novo modeling approaches-AlphaFold2 (AF2), Robetta-RoseTTAFold (Robetta), and transform-restrained Rosetta (trRosetta)-and two template-based tools-the Molecular Operating Environment (MOE) and iterative threading assembly refinement (I-TASSER)-to construct the structure of a viral capsid protein, hepatitis C virus core protein (HCVcp), whose structure have not been fully resolved by laboratory techniques. Templates with sufficient sequence identity for the homology modeling of complete HCVcp are currently unavailable. Therefore, we performed domain-based homology modeling for MOE simulations. The templates for each domain were obtained through sequence-based searches on NCBI and the Protein Data Bank. Then, the modeled domains were assembled to construct the complete structure of HCVcp. The full-length structure and two truncated forms modeled using various computational tools were compared. Molecular dynamics (MD) simulations were performed to refine the structures. The root mean square deviation of backbone atoms, root mean square fluctuation of Cα atoms, and radius of gyration were calculated to monitor structural changes and convergence in the simulations. The model quality was evaluated through ERRAT and phi-psi plot analysis. In terms of the initial prediction for protein modeling, Robetta and trRosetta outperformed AF2. Regarding template-based tools, MOE outperformed I-TASSER. MD simulations resulted in compactly folded protein structures, which were of good quality and theoretically accurate. Thus, the predicted structures of certain proteins must be refined to obtain reliable structural models. MD simulation is a promising tool for this purpose.

Structure of the human T cell leukaemia virus capsid protein – a new drug target

Structure of the human T cell leukaemia virus capsid protein – a new drug target

Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27.

Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation and decreased fertility and induced immunosuppressive disease, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that ribonucleoside-diphosphate reductase subunit M2 (RRM2) plays a critical role during ALV-J infection by interacting with capsid protein P27 and activating Wnt/β-catenin signaling. We found that the expression of RRM2 is effectively increased during ALV-J infection, and that RRM2 facilitates ALV-J replication by interacting with viral capsid protein P27. Furthermore, ALV-J P27 activated Wnt/β-catenin signaling by promoting β-catenin entry into the nucleus, and RRM2 activated Wnt/β-catenin signaling by enhancing its phosphorylation at Ser18 during ALV-J infection. These data suggest that the upregulation of RRM2 expression by ALV-J infection favors viral replication in host cells via activating Wnt/β-catenin signaling. IMPORTANCE Our results revealed a novel mechanism by which RRM2 facilitates ALV-J growth. That is, the upregulation of RRM2 expression by ALV-J infection favors viral replication by interacting with capsid protein P27 and activating Wnt/β-catenin pathway in host cells. Furthermore, the phosphorylation of serine at position 18 of RRM2 was verified to be the important factor regulating the activation of Wnt/β-catenin signaling. This study provides insights for further studies of the molecular mechanism of ALV-J infection.

New viruses of Cladosporium sp. expand considerably the taxonomic structure of Gammapartitivirus genus.

Despite the fact that Cladosporium sp. are ubiquitous fungi, their viromes have been little studied. By analysing a collection of Cladosporium fungi, two new partitiviruses named Cladosporium cladosporioides partitivirus 1 (CcPV1) and Cladosporium cladosporioides partitivirus 2 (CcPV2) co-infecting a strain of Cladosporium cladosporioides were identified. Their complete genome consists of two monocistronic dsRNA segments (RNA1 and RNA2) with a high percentage of pairwise identity on 5' and 3' end. The RNA directed RNA polymerase (RdRp) of both viruses and the capsid protein (CP) of CcPV1 display the classic characteristics required for their assignment to the Gammapartitivirus genus. In contrast, CcPV2 RNA2 encodes for a 41 KDa CP that is unusually smaller when aligned to CPs of other viruses classified in this genus. The structural role of this protein is confirmed by electrophoresis on acrylamide gel of purified viral particles. Despite the low percentage of identity between the capsid proteins of CcPV1 and CcPV2, their three-dimensional structures predicted by AlphaFold2 show strong similarities and confirm functional proximity. Fifteen similar viral sequences of unknown function were annotated using the CcPV2 CP sequence. The phylogeny of the CP was highly consistent with the phylogeny of their corresponding RdRp, supporting the organization of Gammapartitiviruses into three distinct clades despite stretching the current demarcation criteria. It is proposed that a new subgenus be created within the genus Gammapartitivirus for this new group.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Clinical and immunological characteristics for BK polyomavirus-associated nephropathy after kidney transplantation.

BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) can cause a significant risk of allograft impairment after kidney transplantation (KT). Intact BKPyV-specific immunity is associated with viral containment. This study investigated BKPyV-specific immunological factors among KT recipients. This prospective study in a single transplant center from January 2019 to August 2019 assessed associations between clinical and immunological characteristics, with a focus on BKPyV-cell-specific immunity and BKPyVAN, among KT recipients aged ≥15 years. The numbers of interferon-gamma (IFN-γ)-producing CD4+ T, CD8+ T, natural killer (NK), and natural killerT (NKT) cells were measured after stimulation with large T antigen and viral capsid protein 1 (VP1). In total, 100 KT recipients were included (mean age ± SD, 42 ± 11 years); 35% of the recipients were female patients, and 70% had received induction immunosuppressive therapy. The 1-year cumulative incidence of high-level BKPyV DNAuria (possible BKPyVAN) and (presumptive BKPyVAN) was 18%. Among 40 patients with immunological factor data, pre-KT %NK cells (hazard ratio [HR], 1.258; 95% confidence interval [CI], 1.077-1.469; p = .004) and %VP1-specific NK cells (HR, 1.209; 95% CI, 1.055-1.386; p = .006) were factors independently associated with possible and presumptive BKPyVAN. KT recipients with possible and presumptive BKPyVAN were more likely to exhibit significant mean coefficients of %NK, %VP1-specific NK, and %NKT cells at 1 month after KT than before KT (all p < .05). Individuals with nonspecific and VP1-specific NK cells before KT and increasing numbers of these cells after KT may be at risk for high-level BKPyV DNAuria and presumptive BKPyVAN. Further studies are needed to determine the utility of BKPyV-specific innate immune surveillance in predicting the occurrence of BKPyVAN.

Involvement of cutavirus in a subset of patients with cutaneous T-cell lymphoma with an unfavorable outcome

European studies suggest an association between cutavirus (CuV) and cutaneous T-cell lymphoma (CTCL); however, the worldwide prevalence of CuV in patients with CTCL and its prognostic impact remain unknown.We investigated the prevalence and viral loads of CuV DNA using biopsy specimens from the lesional skins of 141 Japanese patients with cutaneous malignancies, including 55 patients with various types of CTCL.CuV DNA was detected significantly more frequently in biopsies from patients with mycosis fungoides (MF) (38% [13/34]; the most common subtype of CTCL) than in those from patients with other cutaneous malignancies (6% [6/107]; P<0.001). The viral-load range in patients with CuV DNA-positive MF was 23-3922 copies/103 cells and 8-65 copies/μg of DNA. A phylogenetic analysis using the partial sequences of the CuV viral capsid protein 1 (VP1)/VP2 genes revealed that the CuV sequences identified here were clustered in a Japanese-specific clade distinct from that comprising CuV sequences from European patients with MF. Kaplan-Meier curves and a log-rank test showed that CuV positivity was associated with a shorter disease-specific survival in patients with MF (P = 0.031), whereas no significant difference in overall survival was observed (P = 0.275). No significant correlation was observed between CuV DNA load and survival in patients with CuV-positive MF.Our results suggest that CuV is associated with MF in a subset of Japanese patients. Large-scale prospective studies are warranted to clarify the role of CuV status, especially regarding the viral genotype, on adverse outcomes in patients with CuV-positive MF.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

Plant virus movement proteins originated from jelly-roll capsid proteins.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus (TMV) MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae. We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

Identification and genetic characteristics of tusavirus in fecal samples of patients with chronic diseases in Guangzhou, China.

The Tunisian stool-associated parvovirus [Tusavirus (TuV)] is a novel member of the genus Protoparvovirus, which may be linked to diarrhea. Herein, we investigated the prevalence of TuV in different populations and analyzed its genetic and bioinformatic characteristics. This study was conducted in a tertiary hospital in Guangzhou (China) from February 2018 to July 2022. Demographic and clinical information and stool samples were collected from individuals who visited the hospital. ProtScale, SwissModel, Datamonkey, and other tools were used to analyze and predict the physicochemical parameters, tertiary structure, selection pressure, and B-cell epitopes of capsid viral protein 2 of TuV (VP2-TuV). A total of 3,837 participants were enrolled, among which two stool samples from patients with chronic illnesses were tested positive for TuV DNA. However, no positive sample was detected among patients with diarrhea. Two near-complete genome sequences were amplified. The genetic analysis revealed the presence of diversity among TuVs isolated from distinct host species. Bioinformatics analysis revealed that VP2-TuV exhibited hydrophilic properties and lacked transmembrane domains and signal peptides. The secondary structure of VP2-TuV was composed mainly of random coils and β-strands. Selective-pressure analysis of the VP2 region suggested that TuV primarily underwent negative selection during evolution. Negatively selected codon sites coincided with residues comprising of B-cell epitopes, suggesting minimal changes in the immunogenicity of TuV over time. TuV was detected in patients with chronic diseases but not in patients with diarrhea. The putative roles of TuV in the pathogenicity of human diseases and zoonotic viruses must be determined by additional studies.

Structural and Biophysical Analysis of Adeno-Associated Virus Serotype 2 Capsid Assembly Variants.

Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability (Tm) than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. IMPORTANCE Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.

Immune response of DNA vaccinated-gilthead seabream (Sparus aurata) against LCDV-Sa infection: relevance of the inflammatory process.

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccine-induced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1β, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.