Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins

Background: Tospoviruses (genus Tospovirus, family Peribunyaviridae, order Bunyavirales) cause significant losses to a wide range of agronomic and horticultural crops worldwide. Identification and characterization of specific sequences and motifs that are critical for virus infection and pathogenicity could provide useful insights and targets for engineering virus resistance that is potentially both broad spectrum and durable. Tomato spotted wilt virus (TSWV), the most prolific member of the group, was used to better understand the structure-function relationships of the nucleocapsid gene (N), and the silencing suppressor gene (NSs), coded by the TSWV small RNA. Methods: Using a global collection of orthotospoviral sequences, several amino acids that were conserved across the genus and the potential location of these conserved amino acid motifs in these proteins was determined. We used state of the art 3D modeling algorithms, MULTICOM-CLUSTER, MULTICOM-CONSTRUCT, MULTICOM-NOVEL, I-TASSER, ROSETTA and CONFOLD to predict the secondary and tertiary structures of the N and the NSs proteins. Results: We identified nine amino acid residues in the N protein among 31 known tospoviral species, and ten amino acid residues in NSs protein among 27 tospoviral species that were conserved across the genus. For the N protein, all three algorithms gave nearly identical tertiary models. While the conserved residues were distributed throughout the protein on a linear scale, at the tertiary level, three residues were consistently located in the coil in all the models. For NSs protein models, there was no agreement among the three algorithms. However, with respect to the localization of the conserved motifs, G was consistently located in coil, while H was localized in the coil in three models. Conclusions: This is the first report of predicting the 3D structure of any tospoviral NSs protein and revealed a consistent location for two of the ten conserved residues. The modelers used gave accurate prediction for N protein allowing the localization of the conserved residues. Results form the basis for further work on the structure-function relationships of tospoviral proteins and could be useful in developing novel virus control strategies targeting the conserved residues. 18 11

Chemical elicitors of systemic acquired resistance—Salicylic acid and its functional analogs

Any interaction of plants with phytopathogens involves the generation of various chemical molecules that are critical for activation of their defense machinery. One of the chemicals, salicylic acid (SA) induces systemic acquired resistance (SAR) in plants. The activation of SAR provides a broad-spectrum resistance against a wide range of related or unrelated pathogens. There has been considerable progress in the biochemical and molecular understanding of SAR activation in various plants. In addition, several chemicals including SA and its analogs are known to provide a direct or indirect defense against pathogens when applied to plants. Molecular mechanism of plant defense induced by synthetic chemical inducers is not very well understood. This review highlights the importance of salicylic acid and its most studied analog, Acibenzolar-S-methyl in inducing SAR and it also provides a description of other major chemical elicitors of plant defenses and their possible molecular mechanism. Keywords: SA, SAR, Acibenzolar-S-methyl, Chemical elicitors, Defense signalin

Movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections

Negative-stranded tospoviruses (family: Bunyaviridae) are among the most agronomically important viruses. Some of the tospoviruses are known to exist as mixed infections in the same host plant. Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV) were used to study virus-virus interaction in dually infected host plants. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors. BiFC results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in dually infected Nicotiana benthamiana plants. BiFC results were further confirmed by pull down and yeast-2-hybrid (Y2H) assays. Interacting regions of the N and NSm proteins were also identified by Y2H system and β-galactosidase activity. Several regions of the N and NSm were found interacting with each other. The regions involved in these interactions are presumed to be critical for the functioning of the tospovirus N and NSm proteins. This is the first report of in vivo protein interactions of distinct tospoviruses in mixed infection

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins.

BACKGROUND:Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS:Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. CONCLUSION/SIGNIFICANCE:Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins

Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes

Occurrence of <i style="">Iris mild mosaic potyvirus</i> in cultivated iris in India

94-98Iris (Iris x hollandica Hort. cv. Bluemagic) plants showing mosaic symptoms were tested for the presence of Iris mild mosaic potyvirus (IMMV) by host range, enzyme linked immunosorbent assay (ELISA) using antibodies specific for IMMV), reverse transcription polymerase chain reaction (RT-PCR), immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) using potyvirus group specific primers and antibodies specific to IMMV, immune electron microscopy and cytopathology. Presence of IMMV was detected by ELISA, confirmed by RT-PCR, IC-RT-PCR, IEM, cytopathology and sequencing of the cloned PCR product. From the sequence of the virus, specific primers were designed and specific detection of IMMV from iris was standardized

Suppression of a Subset of Interferon-Induced Genes by Human Papillomavirus Type 16 E7 via a Cyclin Dependent Kinase 8-Dependent Mechanism

Persistent infection by human papillomaviruses (HPVs), small, double-stranded DNA viruses that infect keratinocytes of the squamous epithelia, can lead to the development of cervical and other cancers. The viral oncoprotein E7 contributes to viral persistence in part by regulating host gene expression through binding host transcriptional regulators, although mechanisms responsible for E7-mediated transcriptional regulation are incompletely understood. Type I IFN signaling promotes the expression of anti-viral genes, called interferon-stimulated genes (ISGs), through the phosphorylation and activation of STAT1. In this study, we have observed that the CR3 domain of E7 contributes to the episomal maintenance of viral genomes. Transcriptome analysis revealed that E7 transcriptionally suppresses a subset of ISGs but not through regulation of STAT1 activation. Instead, we discovered that E7 associates with Mediator kinase CDK8 and this is correlated with the recruitment of CDK8 to ISG promoters and reduced ISG expression. E7 fails to suppress ISGs in the absence of CDK8, indicating that CDK8 function contributes to the suppression of ISGs by E7. Altogether, E7/CDK8 association may be a novel mechanism by which E7 inhibits innate immune signaling

Self-interaction of <i>Iris yellow spot virus</i> (IYSV) nucleocapsid (N) protein.

<p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273), white box, full length <i>Cauliflower mosaic virus</i> P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).</p

Heterotypic interaction of <i>Iris yellow spot virus</i> (IYSV) nucleocapsid (N) and movement (NSm) proteins.

<p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273); yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).</p

List of primers used for bimolecular fluorescence complementation and pull down assays.

<p>^a Primer names with the suffix ‘F’ are forward primers, while those with ‘R’ are reverse primers.</p><p>^b Sequence of primer, where the underlined nucleotides are <i>att</i> sequence for the Gateway cloning.</p><p>List of primers used for bimolecular fluorescence complementation and pull down assays.</p