Structure and assembly of Sesbania mosaic virus

Sesbania mosaic virus (SeMV) is a ss-RNA (4149 nt) plant sobemovirus isolated from farmer's field around Tirupathi, Andhra Pradesh. The viral capsid (30 nm diameter) consists of 180 copies of protein subunits (MW 29 kDa) organized with icosahedral symmetry. In order to understand the mechanism of assembly of SeMV, a large number of deletion and substitution mutants of the coat protein (CP) were constructed. Recombinant SeMV CP (rCP) as well as the N-terminal rCP deletion mutant Delta N22 were found to assemble in E. coli into virus-like particles (VLPs). Delta N36 and Delta N65 mostly formed smaller particles consisting of 60 protein subunits. Although particlem assembly was not affected due to the substitution of aspartates (D14 and D149) that coordinate calcium ions by asparagines, the stability of the resulting capsids was drastically reduced. Deletion of residues forming a characteristic beta-annulus at the icosahedral 3-folds did not affect the assembly of VLPs. Mutation of a single tryptophan, which occurs near the icosahedral fivefold axis to glutamate or lysine, resulted in the disruption of the capsid leading to soluble dimers that resembled the quasi-dimer structure of the native virus. Replacement of positively charged residues in the amino terminal segment of CP resulted in the formation of empty shells. Based on these observations, a plausible mechanism of assembly is proposed.

Host factor Ebp1 inhibits rinderpest virus transcription in vivo

ErbB3 binding protein Ebp1 has been shown to downregulate ErbB3 receptor-mediated signaling to inhibit cell proliferation. Rinderpest virus belongs to the family Paramyxoviridae and is characterized by the presence of a non-segmented negative-sense RNA genome. In this work, we show that rinderpest virus infection of Vero cells leads to the down-regulation of the host factor Ebp1, at both the mRNA and protein levels. Ebp1 protein has been shown to co-localize with viral inclusion bodies in infected cells, and it is packaged into virions, presumably through its interaction with the N protein or the N-RNA itself. Overexpression of Ebp1 inhibits viral transcription and multiplication in infected cells, suggesting that a mutual antagonism operates between host factor Ebp1 and the virus.

Replication of Japanese encephalitis virus in mouse brain induces alterations in lymphocyte response

The experimental model using intracerebral (i.c.) challenge was employed in many studies evaluating the protection against disease induced by Japanese encephalitis virus (JEV). We investigated alterations in peripheral lymphocyte response caused by i.c. infection of mice with JEV. Splenocytes from the i.c.-infected mice showed suppressed proliferative response to concanavalin A (con A) and anti-CD3 antibody stimulation. At the same time, the expression of CD25 (IL-2R) and production of IL-2 was inhibited. Addition of anti-CD28 antibody restored the decreased anti-CD3 antibody-mediated proliferation in the splenocytes. Moreover, the number of con A-stimulated cells secreting IL-4 was significantly reduced in splenocytes from i.c.-infected mice. These studies suggested that the i.c. infection with JEV might involve additional immune modulation effects due to massive virus replication in the brain.

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.

Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants.

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin’s binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy–entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.

Resistance responses blocking systemic movement of Potato virus A in potato

Peruna kestää A-virusta estämällä sen leviämistä Peruna on maissin ohella maailman kolmanneksi tärkein ravintokasvi vehnän ja riisin jälkeen. Perunaa lisätään kasvullisesti mukuloita istuttamalla, jolloin virukset siirtyvät sairaiden siemenmukuloiden välityksellä kasvukaudesta toiseen. Virustauteja voi torjua ainoastaan terveen siemenperunan ja kestävien lajikkeiden avulla. Kestävyys perustuu usein siihen, että kasvi estää viruksen leviämisen tartuntakohdasta välttyäkseen virustaudilta. Tässä työssä tutkittiin kolmea perunan A-viruksen (PVA) liikkumista estävää kestävyysmekanismia perunassa. Lisäksi työn kokeelliseen osaan oleellisesti kuuluvaa virustartutusta varten kehitettiin uusi paranneltu versio geenipyssystä. Tämä itse rakennettu laite optimoitiin PVA:n tartuttamiseen mahdollisimman helposti ja pienin käyttökustannuksin. Tutkimuksen kohteena olleessa perunan risteytysjälkeläistössä oli PVA:ta kestäviä kasveja (ryhmä nnr), jotka estivät viruksen liikkumisen aiheuttamatta oireita tartutuskohdassa, sekä kasveja, joissa PVA aiheutti kuolioläikkinä näkyvän yliherkkyysvasteen (ryhmä HR). Molemmissa kestävyystyypeissä virus pystyi monistumaan ja leviämään solusta soluun paikallisesti, mutta liikkuminen muihin kasvinosiin nilan kautta estyi. Ryhmän nnr kasveissa PVA-tartunta ei aiheuttanut tilastollisesti merkitsevää muutosta useimpien geenien ilmenemiseen tartuntakohdassa. Ainoastaan geeniperhe, joka ilmentää tiettyä proteinaasi-inhibiittoria (PI), reagoi PVA:han 24 tuntia tartutuksesta. Kun tämän PVA:han reagoivan geeniperheen jäsenet hiljennettiin nnr- perunalinjoissa, ne muuttuivat alttiiksi PVA:lle ja virus levisi tartuntakohdasta muihin kasvinosiin. Tulos osoittaa, että PI on viruskestävyystekijä. Lisäksi muut tutkimuksessa saadut tulokset tukevat mahdollisuutta, että PI estää PVA:n P1-proteinaasin toimintaa. HR-linjoissa todettiin erilaisiin puolustusvasteisiin liittyvien PR-geenien aktivoitumista PVA-tartunnan seurauksena, mutta myös ilman sitä kasvien kasvettua mullassa noin neljä viikkoa. Sen sijaan solukkoviljelyssä tai vasta kaksi viikkoa mullassa kasvaneissa kasveissa vastaavaa ei vielä todettu. Tulos viittaa siihen, että HR-perunat reagoivat herkemmin ympäristöön ja/tai kasvin kehitysasteeseen laukaisten puolustusvasteita, jotka saattavat parantaa kestävyyttä taudinaiheuttajia vastaan. Kolmas tutkittu kestävyystyyppi havaittiin Pito-perunalajikkeessa. Se muistutti nnr-kestävyyttä siten, että myös siinä viruksen liikkuminen nilassa muihin kasvinosiin estyi. PVA:n todettiin pysähtyvän vasta lehtiruodin tyvelle muodostuvaan irtoamisvyöhykkeeseen, mitä havainnollistettiin käyttämällä muunnettua PVA-rotua, joka tuotti UV-valossa fluoresoivaa vihreää valoa. Tulos viittaa siihen, että virus ei pääse kulkemaan vyöhykkeeseen kuuluvan suojaavan kerroksen läpi, jollei sillä ole pääsyä nilaan. Tällainen kestävyys on tarpeen, jotta virus ei voi korvata nilakuljetusta solusta soluun leviämisellä. Tulokset tuovat uusia näkökulmia kasvien viruskestävyyteen ja auttavat selittämään viruksen nilakuljetuksen estymistä sekä solusta soluun leviämisen pysähtymistä kestävissä kasveissa.

Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93-102 aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type 0 FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153 aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at N-terminal half (1-75 aa) but exhibited variability or substitutions towards C-terminal region (80-153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment. (C) 2010 Elsevier B.V. All rights reserved.

Effective treatment of infectious diseases: A nonlinear adaptive control theoretic approach

A nonlinear adaptive system theoretic approach is presented in this paper for effective treatment of infectious diseases that affect various organs of the human body. The generic model used does not represent any specific disease. However, it mimics the generic immunological dynamics of the human body under pathological attack, including the response to external drugs. From a system theoretic point of view, drugs can be interpreted as control inputs. Assuming a set of nominal parameters in the mathematical model, first a nonlinear controller is designed based on the principle of dynamic inversion. This treatment strategy was found to be effective in completely curing "nominal patients". However, in some cases it is ineffective in curing "realistic patients". This leads to serious (sometimes fatal) damage to the affected organ. To make the drug dosage design more effective, a model-following neuro-adaptive control design is carried out using neural networks, which are trained (adapted) online. From simulation studies, this adaptive controller is found to be effective in killing the invading microbes and healing the damaged organ even in the presence of parameter uncertainties and continuing pathogen attack.

Primary structure of belladonna mottle virus coat protein.

The coat protein of belladonna mottle virus (a tymovirus) was cleaved by trypsin and chymotrypsin, and the peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase, and ion pair chromatography. The peptides were sequenced manually using the 4-N, N-dimethylaminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double-coupling method. The chymotryptic peptides were aligned by overlapping sequences of tryptic peptides and by homology with another tymovirus, eggplant mosaic virus. The belladonna mottle virus is more closely related to eggplant mosaic virus than to turnip yellow mosaic virus, the type member of this group, as evident from the sequence homologies of 57 and 32%, respectively. The accumulation of basic residues at the amino terminus implicated in RNA-protein interactions in many spherical plant viruses was absent in all the three sequences. Interestingly, the amino-terminal region is the least conserved among the tymoviruses. The longest stretch of conserved sequence between belladonna mottle virus and eggplant mosaic virus was residues 34-44, whereas it was residues 96-102 in the case of belladonna mottle virus and turnip yellow mosaic virus. A tetrapeptide in the region (residues 154-157) was found to be common for all the three sequences. It is possible that these conserved regions (residues 34-44, 96-102, 154-157) are involved in either intersubunit or RNA-protein interactions.

Characterization of foot-and-mouth disease virus types Ο and Asia 1 RNA

Foot-and-mouth disease is an acute and highly contagious febrile disease affecting cloven-footed animals. Identification of the foot-and-mouth disease virus (FMDV), the causative agent of the disease, posed problems because of the occurrence of many types and subtypes of the virus. A molecular approach based on oligonucleotide mapping of FMDV RNA has been used for the identification and characterization of virus isolates obtained in a disease outbreak (King et al., 1981). One-dimensional oligonucleotide mapping was used for rapid analysis of FMDV RNA (LaTorre et al., 1982). FMDV types Ο and Asia 1 of Indian origin are being routinely used for vaccine production in India. This report presents the differences between FMDV types Ο and Asia 1 at molecular level based on one-dimensional oligonucleotide mapping of virus-induced poly (A) RNA.