VP1 Mutants Research Articles

CD4 T cell deficiency narrows virus-neutralizing IgG coverage for Polyomavirus and selects for antibody-escape variants

Abstract CD4 T cell deficiencies predispose patients to progressive multifocal leukoencephalopathy (PML), a brain disease caused by JC polyomavirus (JCPyV). PML is linked to immunomodulatory therapies that preferentially lower CD4 T cells in the CSF (natalizumab); but how CD4 T cells control JCPyV is undefined. “Blind spots” in the ability to neutralize JCPyV variants with mutations in the VP1 capsid protein were described in PML patients. We recently reported that a VP1 mAb selects for VP1-mutant escape variants both in vitroand in vivo; some of these variant viruses are neurovirulent. We hypothesized that alterations in the Ab response due to CD4 T cell insufficiency underlie emergence of Ab-escape mouse polyomavirus (MuPyV) variants. T-cell independent (TI) VP1-specific IgG (via anti-CD4 mAb depletion, CD40L blockade, MHCII KO mice, IL21R KO mice) neutralizes MuPyV but has reduced titer and avidity than anti-VP1 IgGs in CD4 T cell-sufficient mice. Serial passaging of MuPyV in vitrowith sera from TI mice selected an Ab-escape VP1 mutant containing amino acid deletions at residue 145–146 in an external loop of VP1 (MuPyV.145–146). In contrast to sera from healthy MuPyV-infected mice, TI sera failed to neutralize MuPyV.145–146. Depletion of CD4 T cells during persistent MuPyV infection, as a model of acquired CD4 T cell insufficiency (i.e. onset of immunomodulatory mAbs) via anti-CD4 mAb depletion, CD40L blockade, and anti-CD20 mAb B cell depletion also reduced anti-VP1 IgG titer and avidity. Ongoing studies seek to determine if a similar loss in recognition of the 145–146 VP1 epitope occurs. Our data shows that a narrowed antiviral IgG response due to CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses. Supported by grants from the NIH (5R01NS088367, 5R01NS092662, and R35NS127217) (AEL) Judy S. Finkelstein Memorial Award (KNA) T32 CA06395 (KNA)

Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing.

AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

CD4 T cell deficiency results in impaired neutralizing IgG against polyomavirus carrying a frequent PML-associated capsid mutation

Abstract Deficiencies in the CD4 T cell compartment, such as HIV/AIDS, idiopathic CD4 lymphopenia, predispose patients to progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease caused by the JC polyomavirus (JCPyV). PML is associated with immunomodulatory therapies that preferentially diminish CD4 T cells in the CSF (natalizumab) or B cells (rituximab). The connection between CD4 T cells and JCPyV control is unclear. Work by others show that PML patient sera have “blind spots” in neutralizing JCPyV variants with mutations in the VP1 capsid protein. We hypothesized that defects in the humoral response after CD4 T cell depletion led to the emergence of antibody (Ab)-escape PyV variants. Using mouse PyV (MuPyV) to model JCPyV CNS disease, we found immune cell aggregates consisting of B cells, CD4 T cells, and CD8 T cells along the ventricles of PyV-infected WT mice but only a few infected cells. In contrast, CD4 T cell-depleted mice had more infected ependyma but lacked lymphocytic aggregates. There were fewer anti-PyV Ab-secreting B cells in the CNS despite similar B cell numbers in MuPyV-infected WT and CD4 T cell deficient mice brains. CD4 T cells prevent emergence of VP1 mutant PyVs that escape neutralizing Abs. MuPyV infection elicits a CD4 T cell-independent anti-VP1 IgG response that neutralizes WT virus equivalently to IgG from CD4 T cell-sufficient mice. Serum from MHC-II KO and CD4 mAb depleted mice had significantly lower neutralization activity toward a MuPyV with a frequent PML-equivalent VP1 mutation than to WT MuPyV. This data demonstrate that a narrowed antiviral IgG response caused by CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses. Supported by 1. Finkelstein Memorial Student Research Award, Pennsylvania State University College of Medicine, Hershey, PA 2. T32 CA060395 Training Grant (Meyers), NIH/NCI 3. R01 NS088367, NIH/NCI

A Novel Core Effector Vp1 Promotes Fungal Colonization and Virulence of Ustilago maydis

The biotrophic fungus Ustilago maydis secretes a plethora of uncharacterized effector proteins and causes smut disease in maize. Among the effector genes that are up-regulated during the biotrophic growth in maize, we identified vp1 (virulence promoting 1), which has an expression that was up-regulated and maintained at a high level throughout the life cycle of the fungus. We characterized Vp1 by applying in silico analysis, reverse genetics, phenotypic assessment, microscopy, and protein localization and provided a fundamental understanding of the Vp1 protein in U. maydis. The reduction in fungal virulence and colonization in the vp1 mutant suggests the virulence-promoting function of Vp1. The deletion studies on the NLS (nuclear localization signal) sequence and the protein localization study revealed that the C-terminus of Vp1 is processed after secretion in plant apoplast and could localize to the plant nucleus. The Ustilago hordei ortholog UhVp1 lacks NLS localized in the plant cytoplasm, suggesting that the orthologs might have a distinct subcellular localization. Further complementation studies of the Vp1 orthologs in related smut fungi revealed that none of them could complement the virulence function of U. maydis Vp1, suggesting that UmVp1 could acquire a specialized function via sequence divergence.

Persistent BK Polyomavirus Viruria is Associated with Accumulation of VP1 Mutations and Neutralization Escape.

To investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific “holes” in the patient’s humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies.

Evidence of combined effect of amino acid substitutions within G-H and B-C loops of VP1 conferring serological heterogeneity in foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) serotype A exhibits a higher degree of genetic and antigenic diversity resulting in frequent vaccine failure due to serological mismatch between the vaccine and heterologous strains. Currently, knowledge on the molecular basis of antigenic relationships among the FMDVs is limited; nevertheless, intratype antigenic variation due to mutation(s) is widely considered as the main hurdle to appropriate FMD vaccine development. Here, we studied genetic and antigenic variations of four FMDV serotype A isolates, BAN/GA/Sa-197/2013 (BAN-197), BAN/CH/Sa-304/2016 (BAN-304), BAN/DH/Sa-307/2016 (BAN-307) and BAN/DH/Sa-310/2017 (BAN-310) circulating in Bangladesh during 2013-2017. Initially, antigenic relationships (r1 -values) of the field isolates were evaluated by the two-dimensional microneutralization test (2D-MNT) using the hyperimmune antisera raised in cattle against the vaccine strain, BAN-304. Interesingly, the results showed protective serological cross-reactivity (r1 -values>0.4) between the vaccine strain and the field isolates, BAN-307 and BAN-310, except BAN-197 that substantially mismatched (r1 =0.129±0.043) with the BAN-304. Although VP1-based phylogeny grouped all the isolates within the same sublineage C (a subgroup of VP3Δ59 variant) under the lineage A/ASIA/G-VII, strikingly, computational analyses of the viral capsid proteins demonstrated significant deviation at the VP1 G-H loop of BAN-197 from the vaccine strain, while VP(2-4) of both isolates were structurally conserved. To bridge the gap of how the distortion of the G-H loop and consequent antigenic hetergeneity occurred in BAN-197, we performed in silico combinatorial substitutions of the VP1 mutant amino acids (aa) of BAN-197 with the respective residues in BAN-304. Remarkably, our analyses revealed that two substitutions of distantly located aa at B-C (T48I:threonine→isoleucine) and G-H (A143V:alanine→valine) loops, in combination, distorted the VP1 G-H loop. Overall, this work contributes to understanding the molecular basis of antigenic relationships operating in serotype A FMDVs and the selection of suitable vaccine strain(s) for effective prophylaxis of FMD based on VP1-based analyses.

The surface-exposed loop region of norovirus GII.3 VP1 plays an essential role in binding histo-blood group antigens.

Trypsin digestion promotes disassembly of GII.3 NoV virus-like particles (VLPs) and binding of VLPs to salivary histo-blood group antigens (HBGAs), but it is not clear which specific regions or residues mediate viral attachment to HBGAs. An earlier study indicated that arginine residues in the predicted surface-exposed loop region are susceptible to trypsin digestion. Here, we introduced single or multiple substitutions of four arginine residues located in the predicted surface-exposed loop region of the GII.3 NoV capsid protein (VP1) and observed their effects on susceptibility to trypsin digestion and binding to HBGAs. All of the mutations in VP1, including single substitutions (R287G, R292G, R296G or R307G) and quadruple substitutions (R287G, R292G, R296G and R307G), permitted successful VLP assembly. After tryptic digestion, all VP1 proteins bearing single point mutations were cleaved, resulting in complete digestion or single fragments with various molecular sizes (27-35kDa), while the VP1 protein bearing four substitutions was cleaved into two fragments (27-55kDa). Binding assays using synthetic and salivary HBGAs showed that none of the VP1 mutants (singly or quadruply substituted) exhibited detectable binding to HBGA before or after trypsin cleavage. These results indicated that arginine residues within the predicted surface loop region of GII.3 NoV VP1 were involved directly or indirectly in binding salivary HBGAs and could potentially mediate the HBGA-GII.3 NoV interactions through which host cells become infected.

Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation.

ABSTRACTSimian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40.

Up-regulation of epithelial Na+ channel ENaC by human parvovirus B19 capsid protein VP1

BackgroundClinical disorders caused by parvovirus B19 (B19V) infection include endothelial dysfunction with cardiac ischemia. The virus is effective in part by lysophosphatidylcholine-producing phospholipase A2 (PLA2) activity of B19V capsid protein VP1. Mechanisms compromising endothelial function include up-regulation of amiloride sensitive epithelial Na+-channel ENaC leading to endothelial cell stiffness. Regulators of ENaC include ubiquitin-ligase Nedd4-2. The present study explored whether VP1 modifies ENaC-activity. MethodscRNA encoding ENaC was injected into Xenopus oocytes without or with cRNA encoding VP1. Experiments were made with or without coexpression of Nedd4-2. ENaC activity was estimated from amiloride (50 μM) sensitive current. ResultsInjection of cRNA encoding ENaC into Xenopus oocytes was followed by appearance of amiloride sensitive current, which was significantly enhanced by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on ENaC was mimicked by treatment of ENaC expressing oocytes with lysophosphatidylcholine (1 μg/ml). The effect of VP1 and lysophosphatidylcholine was not additive. ENaC activity was downregulated by Nedd4-2, an effect not reversed by VP1. ConclusionsThe B19V capsid protein VP1 up-regulates ENaC, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.

Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops.

Down-regulation of inwardly rectifying Kir2.1 K+ channels by human parvovirus B19 capsid protein VP1.

Parvovirus B19 (B19V) has previously been shown to cause endothelial dysfunction. B19V capsid protein VP1 harbors a lysophosphatidylcholine producing phospholipase A2 (PLA2). Lysophosphatidylcholine inhibits Na(+)/K(+) ATPase, which in turn may impact on the activity of inwardly rectifying K(+) channels. The present study explored whether VP1 modifies the activity of Kir2.1 K(+) channels. cRNA encoding Kir2.1 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced inflammatory cardiomyopathy or the VP1 mutant (H153A)VP1 lacking a functional PLA2 activity. K(+) channel activity was determined by dual electrode voltage clamp. In addition, Na(+)/K(+)-ATPase activity was estimated from K(+)-induced pump current (I(pump)) and ouabain-inhibited current (I(ouabain)). Injection of cRNA encoding Kir2.1 into Xenopus oocytes was followed by appearance of inwardly rectifying K(+) channel activity (I(K)), which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding (H153A)VP1. The effect of VP1 on I K was mimicked by lysophosphatidylcholine (1 μg/ml) and by inhibition of Na(+)/K(+)-ATPase with 0.1 mM ouabain. In the presence of lysophosphatidylcholine, I K was not further decreased by additional treatment with ouabain. The B19V capsid protein VP1 thus inhibits Kir2.1 channels, an effect at least partially due to PLA2-dependent formation of lysophosphatidylcholine with subsequent inhibition of Na(+)/K(+)-ATPase activity.

Down-regulation of K+ channels by human parvovirus B19 capsid protein VP1

Parvovirus B19 (B19V) can cause inflammatory cardiomyopathy and endothelial dysfunction. Pathophysiological mechanisms involved include lysophosphatidylcholine producing phospholipase A2 (PLA2) activity of the B19V capsid protein VP1. Most recently, VP1 and lysophosphatidylcholine have been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies the activity of Kv1.3 and Kv1.5 K+ channels.cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced myocarditis. K+ channel activity was determined by dual electrode voltage clamp.Injection of cRNA encoding Kv1.3 or Kv1.5 into Xenopus oocytes was followed by appearance of Kv K+ channel activity, which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on Kv current was not significantly modified by transcription inhibitor actinomycin (10μM for 36h) but was mimicked by lysophosphatidylcholine (1μg/ml).The B19V capsid protein VP1 inhibits host cell Kv channels, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.

Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

Distinct functions of COAR and B3 domains of maize VP1 in induction of ectopic gene expression and plant developmental phenotypes in Arabidopsis

Arabidopsis ABI3 and maize VP1 are orthologous transcription factors that regulate seed maturation. ABI3 and VP1 have a C-terminal B3 DNA binding domain and a conserved N-terminal co-activator/co-repressor (COAR) domain consisting of A1, B1, B2 sub-domains. The COAR domain mediates abscisic acid signaling via a physical interaction with ABI5-related bZIP proteins. In order to delineate the COAR and B3 domain dependent functions of VP1, we created site directed mutations in the B3 domain that disrupted DNA binding activity and characterized gene regulation by the mutant proteins in transgenic abi3 mutant Arabidopsis plants. In seeds, COAR domain function of VP1 mutants that lacked B3 DNA binding activity was sufficient for complementation of the desiccation intolerant seed phenotype of abi3. Similarly in seedlings, the B3 domain was dispensable for most VP1 induced gene expression and ectopic developmental phenotypes, except for a small subset of the genes that showed B3 dependent regulation. Unexpectedly, over-expression of the DNA-binding deficient VP1-K519R mutant protein caused quantitative changes in floral organ size including elongation of pistils and shortened stamen filaments that resulted in a self-incompatible longistyly flower morphology, a key component of heterostyly type self-incompatibility.

An Increase in Acid Resistance of Foot-and-Mouth Disease Virus Capsid Is Mediated by a Tyrosine Replacement of the VP2 Histidine Previously Associated with VP0 Cleavage

The foot-and-mouth disease virus (FMDV) capsid is highly acid labile, but introduction of amino acid replacements, including an N17D change in VP1, can increase its acid resistance. Using mutant VP1 N17D as a starting point, we isolated a virus with higher acid resistance carrying an additional replacement, VP2 H145Y, in a residue highly conserved among picornaviruses, which has been proposed to be responsible for VP0 cleavage. This mutant provides an example of the multifunctionality of picornavirus capsid residues.

Insights into Stabilization of a Viral Protein Cage in Templating Complex Nanoarchitectures: Roles of Disulfide Bonds

As a typical protein nanostructure, virus-based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter-pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)-containing VNPs (VNP-QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP-QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein-based functional nanostructures.

Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation.

Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations. A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant). Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na⁺/K⁺ ATPase. The present study explored whether VP1 modifies Na⁺/K⁺ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K⁺ induced pump current (I_pump) as well as ouabain-inhibited current (I_ouabain) both reflecting Na⁺/K⁺-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, I_pump and I_ouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased I_pump in human microvascular endothelial cells (HMEC). Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na⁺/K⁺ ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine.

Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket

The rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly.

BK virus‐associated infection in cerebrospinal fluid of neurological patients and mutation analysis of the complete VP1 gene in different patient groups

While BK virus (BKV) is frequently associated with pathological conditions in bone marrow and renal transplant recipients, BKV infection in neurological individuals has been rarely reported. As a result of a BKV, JCV, and SV40 large T antigen-specific multiplex PCR on 2,062 cerebrospinal fluid (CSF) samples from neurological patients suspicious of JCV infection, we identified 20 subjects with at least 1 CSF specimen positive for BKV large T antigen DNA. Because VP1 protein has been suggested to influence the biological/pathological properties of BKV, we tried to sequence the entire VP1 gene in the BKV-positive neurological patients and succeeded in 14 of the 20 neurological patients. To compare the VP1 sequence of the BKV neurological strains with that of non-neurotropic strains in other clinical situations, full-length VP1 DNA was sequenced in 15 renal and 6 bone marrow transplant recipients positive to BKV-viremia, and in 8 pregnant women as non-pathological controls. An increased (respectively, decreased) tendency for mutations in the BC loop (respectively, EF loop) was observed, and no mutations were detected in the CD, GH, and HI loops. Subtype I was predominant (93%) and compared to archetypal BKV (WW), amino acid substitutions were detected in 4/14 neurological patients, 10/15 renal transplant recipients, 3/6 bone marrow transplant patients, and in all the pregnant women. Each patient group had distinctive VP1 mutations, but these unique substitutions were not present in all patients of this group. However, molecular modeling simulations of the VP1 mutants predicted changes in protein surface properties which might affect the VP1-receptor interaction.