NSs Encoded by Groundnut Bud Necrosis Virus Is a ifunctional Enzyme

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV-tomato (Karnataka) [1] was over-expressed in E.coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5' (beta, gamma imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP.Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5' RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5' alpha phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5' alpha phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5'phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Similarities in the Genomic Sequence and Coat Protein-Structure of Plant Viruses

The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein of three biologically distinct isometric plant viruses, tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard β-barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed.

GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

Plant viruses exploit the host machinery for targeting the viral genome-movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein la (PDLP la) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER-GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER-GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130-138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm interacts with NP via its N-terminal unfolded region and the NSm-NP complex could in turn interact with the ER membrane via the C-terminal coiled coil domain of NSm to form vesicles that are targeted to PD and there by assist the cell to cell movement of the viral genome complex. (C) 2015 Elsevier Inc. All rights reserved.

A novel adeno-associated virus serotype (AAV)-8 capsid mutant improves human coagulation factor IX expression in vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising ``multiantigen'' vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Immunogenic and protective properties of haemagglutinin protein (H) of Rinderpest virus expressed by a recombinant baculovirus

The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant buculovirus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.

Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV: Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.

High-resolution refinement of orthorhombic bovine pancreatic phospholipase A2

The X-ray structure of recombinant bovine pancreatic phospholipase A(2) (PLA2), which specifically catalyzes the cleavage of the sn-2 acylester bond of phospholipids, has been refined at 1.5 Angstrom resolution. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a = 47.12, b = 64.59 and c = 38.14 Angstrom similar to the native enzyme reported previously by Dijkstra et nl. [J. Mel. Biol. (1981), 147, 97-123]. The refinement converged to an R value of 18.4% (R-free = 22.8%) for 16 374 reflections between 10.0 and 1.5 Angstrom resolution. The surface-loop residues (60-70) art: ordered in the present orthorhombic recombinant enzyme, but disordered in the trigonal recombinant enzyme. The active-site residues, His48, Asp99, and the catalytic water superimpose well with the trigonal form. Besides the catalytic water which is hydrogen bonded to His48, it is often seen that there is a second water attached to the same N atom of His48 and simultaneously hydrogen bonded to the O atom of Asp49. It is thought that the second water facilitates the tautomerism of His48 for enzyme catalysis, The catalytic water is also hydrogen bonded to the equatorial water coordinated to the calcium ion, In addition to the equatorial water, there is also an axial calcium water and the additional structural water. These five common water molecules are hydrogen bonded to the additional 16 water molecules in the present orthorhombic structure which may further enhance the structural integrity of the active site. Besides the protein and one calcium ion, a total of 134 water molecules were located in the present high-resolution refinement.

Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus

Japanese encephalitis virus (JEV) envelope (E) protein has been shown to play a critical role in attachment to cells. However, the receptor interacting with envelope protein has not been conclusively identified. Using mouse neuroblastoma (Neuro2a) cells and purified JEV-E protein in `Virus Overlay Protein Binding Assay' followed by MALDI-TOF analysis, we identified `heat shock protein 70' (Hsp70) as a possible receptor for JEV. Indirect immunofluorescence and flow-cytometry analysis demonstrated localization of Hsp70 on Neuro2a cell surface. Co-immunoprecipitation followed by Western blot analysis reconfirmed the interaction between Hsp70 and JEV-E protein. Further, anti-Hsp70 polyclonal-antibodies were able to block JEV entry into Neuro2a cells. Additionally, using the bioinformatic tool - FTDOCK, clocking between the proteins was performed. Amongst six interacting structural poses studied one pose involving RGD motif on JEV-E and leucine(539) on Hsp70 displayed stable interaction. These observations indicate that Hsp70 serves as putative receptor for JEV in Neuro2A cells.

Inhibition of avian myeloblastosis virus reverse transcriptase and virus inactivation by metal complexes of isonicotinic acid hydrazide

The cupric and ferric complexes of isonicotinic acid hydrazide (INH) inhibit the DNA synthesis catalysed by avian myeloblastosis virus (AMV) reverse transcriptase. The inhibition was to the extent of 95% by 50 μM of cupric-INH complex and 55% by 100 μM of ferric-INH complex. These complexes have been found to bind preferentially to the enzyme than to the template-primer. Kinetic analysis showed that the cupric-INH complex is a non-competitive inhibitor with respect to dTTP. The time course of inhibition has revealed that the complexes are inhibitory even after the initiation of polynucleotide synthesis. In vivo toxicity studies in 1-day-old chicks have shown that the complexes are not toxic up to a concentration of 500 μg per chick. Infection of the 1-day-old chicks with AMV pretreated with 150 μg of either of the complexes prevented symptoms of leukemia due to virus inactivation.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (L-pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups - Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (<5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or onvergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L-pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the L-pro (P<0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P<0.01; 0.003**).