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

Peer reviewe

Processing of SeMV polyproteins revisited

Processing of Sesbania mosaic virus (SeMV) polyprotein 2a and 2ab was reanalyzed in the view of the new genome organization of sobemoviruses. Polyprotein 2a when expressed in E coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8, respectively. Additionally, a novel cleavage was identified within the protease domain at position E132-S133, which was found to be essential for efficient polyprotein processing. Products, corresponding to cleavages identified in E. coli, were also detected in infected Sesbania leaves. Interestingly, though the sites are exactly the same in polyprotein 2ab, it got cleaved between Protease-VPg but not between VPg-RdRp. This indicates to a differential cleavage preference, governed probably by the conformation of 2ab. Also, the studies revealed that, in SeMV, processing is regulated by mode of cleavage and context of the cleavage site

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

Determination of 3' -terminal nucleotide sequence of pepper break vein banding virus RNA and expression of its coat protein in Escherichia coli

Pepper vein banding virus (PVBV) is an important virus infecting chilli pepper in south India. Earlier reports suggested it to be a distinct potyvirus. The nucleotide sequence of PVBV RNA from the 3-end (3862 nt) was determined. Analysis of the nucleotide and deduced amino acid sequence revealed that it encompasses a partial open reading frame encoding the partial sequence of VPE, NIa-protease, NIb, coat protein (CP) and 3-untranslated region (UTR). Comparison of the amino acid sequence of CP and the nucleotide sequence of 3-UTR with those of other potyviruses confirmed an earlier observation that PVBV is a distinct member of the Potyvirus sub-group and it had significant similarity to a recently characterized virus infecting chilli pepper, chilli vein- banding mottle virus (CVbMV), from Thailand. The analysis showed that both PVBV and CVbMV might represent strains of the same virus. Further, the PVBV CP gene was overexpressed in E. coli, which assembled into potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate the CP mRNA

Mutational analysis of the NIa protease from pepper vein banding potyvirus

The nuclear inclusion protein a (NIa) protease plays an important role in the life cycle of potyviruses by processing the viral polyprotein into functional proteins. For functional characterization, the NIa protease from Pepper vein banding potyvirus (PVBV) was overexpressed in Escherichia coli and purified. Using a recombinant polyprotein substrate containing the nuclear inclusion protein b (NIb)-coat protein (CP) cleavage site, a trans-cleavage assay was developed for the NIa protease. The polyprotein substrate also possessed the cleavage site between NIa and NIb, in addition to the NIb-CP site. However, no trans-cleavage by the NIa protease between NIa and NIb was detected indicating that the cleavage between NIa and NIb under natural conditions would be by a cis-cleavage reaction. Site-specific mutations of the conserved residues D81, D90, C110, T146, C151 and H167 were performed to investigate their roles in the catalytic process of the protease. Such an analysis has revealed that D81 and C151 constitute two of the catalytic triad residues in the NIa protease, D90 and C110 are not essential for catalysis, and T146 and H167 are probably involved in binding to Gln at the P1 position of the substrate

Characterization of RNA dependent RNA polymerase (RdRp) of Sesbania mosaic virus a member of genus Sobemovirus

Sesbania mosaic virus (SeMV) belongs to the genus sobemovirus and it infects sesbania grandiflora pers agathi. SeMV is a single stranded positive sense RNA virus, with 5\’ end of the genome covalently-linked viral protein called VPg (viral protein genome linked). The 3\’ end of the genomic RNA lacks a poly (A) tail and does not have t-RNA like structure. The genome of SeMV has three overlapping frames, a small ORF1 at 5\’ end (movement protein) and ORF3 at 3\’ terminus (coat protein). The central part of the genome has two overlapping ORFs, ORF2a and ORF2ab both coding for polyproteins, protease –Vpg-p10-p8 and protease-Vpg-RdRp respectively. ORF2b (RdRp) is expressed by -1 ribosomal frameshifting mechanism. Replication of sobemoviruses and the role of cis- and transacting elements are poorly understood. In order to elucidate the mechanism of replication, we cloned and overexpressed RdRp of SeMV in E. coli. His-tagged full length RdRp was found to be insoluble. Efforts to make the protein soluble under different conditions and by deletion of C-terminal hydrophobic residues were unsuccessful. Therefore RdRp was cloned as GST fusion protein. GST RdRp was soluble however; the purification was hampered by its inability to bind GSH beads. Hence, total crude E.coli lysate expressing GST-RdRp was used for functional analysis. We observed that the GST-RdRp was functionally active and it requires genomic RNA as template for polymerase function

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous genomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication

In vitro expression of belladonna mottle virus genome

In vitro translation of belladonna mottle virus BDMV(I) genomic RNA in a rabbit reticulocyte lysate system produced proteins of Mr 210,000, 150,000 and 78,000 which form the non-structural proteins. The coat protein, on the other hand, was expressed from a subgenomic RNA which was found to be encapsidated in the empty capsids forming the top component viral particles. The implications of subgenomic RNA encapsidation in viral replication and assembly are discussed