Sesbanian mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication

Peer reviewe

Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype

Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking

Lipid Polarization during Cytokinesis

The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp–p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, CΔ43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp

<em>Sesbania Mosaic Virus</em> (SeMV) Infectious Clone: Possible Mechanism of 3′ and 5′ End Repair and Role of Polyprotein Processing in Viral Replication

<div><p><em>Sesbania mosaic virus</em> (SeMV) is a positive stranded RNA virus belonging to the genus <em>Sobemovirus</em>. Construction of an infectious clone is an essential step for deciphering the virus gene functions <em>in vivo</em>. Using <em>Agrobacterium</em> based transient expression system we show that SeMV icDNA is infectious on <em>Sesbania grandiflora</em> and <em>Cyamopsis tetragonoloba</em> plants. The efficiency of icDNA infection was found to be significantly high on <em>Cyamopsis</em> plants when compared to that on <em>Sesbania grandiflora</em>. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3′ and 5′ terminal deletion mutants, we propose a possible mechanism for 3′ and 5′ end repair <em>in vivo</em>. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in <em>trans</em>. However, the <em>trans</em> acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.</p> </div

Description of oligonucleotides used in the study.

<p>Description of oligonucleotides used in the study.</p

Northern analysis of SeMV icDNA infiltrated cotyledon leaves of <i>Cyamopsis tetragonoloba</i> plants.

<p>(a) 0.8% TBE agarose gel (EtBr staining) and northern blot analysis of total RNA extracted from SeMV icDNA agroinfiltrated leaves 6 dpi from three different plants. The negative sense ³²P labelled probe used for hybridization was complementary in sequence to the (+) sgRNA. (b) 0.8% TBE agarose gel analysis (EtBr staining) and northern blot analysis of total RNA extracted from SeMV icDNA agroinfiltrated leaves 6 dpi. The positive sense ³²P labelled probe corresponding in sequence to that of (+) sgRNA was used for hybridization.</p