Determination of the structure of the recombinant T = 1 capsid of Sesbania mosaic virus

The recombinant coat protein (CP) of Sesbania mosaic virus lacking segments of different lengths from the N-terminus expressed in E. coli was shown to selfassemble into a variety of distinct capsids encapsidating 23S rRNA from the host and CP mRNA in vivo. Particles with 60 copies (T = 1) of protein subunits were observed when protein lacking 65 amino acids from the N-terminus was expressed. This recombinant protein possesses the sequence corresponding to the S-domain of the native, T = 3 icosahedral particles but lacks the β-annulus, the βA strand (residues 67–70) and the arginine-rich ARM motif (residues 28–36). Purified T = 1 particles crystallized in the monoclinic space group P21 with cell parameters of a = 188.4 Å, b = 194.6Å, c = 272.1Å and β=92.6°. The structure of the T = 1 particles was determined by X-ray diffraction at 3.0 Å resolution. As expected, the poly-peptide fold of the subunit closely resembles that of the S-domain of the native virus. The recombinant particles bind calcium ions in a manner indistinguishable from that of the native capsids. The structure reveals the major differences in the quaternary organization responsible for the formation of T = 1 against T = 3 particles

Characterization of a Newly Identified 35-Amino-Acid Component of the Vaccinia Virus Entry/Fusion Complex Conserved in All Chordopoxviruses▿

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 Å using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the ΔN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus ∗

Summary. The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73 % identity). The 5 ′ non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of Mr 18370 has only 34 % identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78 % identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326–337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E 325-T 326. The cleavage sit

The role of arginine-rich motif and β-annulus in the assembly and stability of Sesbania mosaic virus capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by protein–protein, protein–RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the β-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein–protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of β-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the ß-annulus to alanine, or deletion of residues 48–53 involved in hydrogen bonding interactions with residues 54–58 of the 3-fold related subunit or deletion of all the residues (48–59) involved in the formation of ß-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the β-annulus. The ordered ß-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions

Structure and function of Sesbania mosaic virus serine protease domain

Poly-protein processing is a common strategy used by many viruses to generate different functional products from a single protein. Viral proteases play a crucial role in this maturation process. Sesbania mosaic virus (SeMV) polyprotein was shown to undergo proteolytic processing when expressed in E. coli. Mutational analysis of the proposed catalytic triad residues (H181, D216 and S284) present in the N-terminal serine protease domain of the polyprotein showed that the protease was indeed responsible for this processing. Analysis of the cleavage site mutants confirmed the cleavage between Protease-VPg, VPg-P10 and P10-P8 at E{325}T{326}, E {402}T{403}and E{498}S{499}sites, respectively. Thus, the protease has both E-T and E-S specificities. The polyprotein has a domain arrangement of Protease-VPg-P10-P8, which is cleaved by the protease. The purified serine protease was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at its C-terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation and VPg resembled "natively unfolded" proteins. CD spectral analysis of DN70Pro-VPg fusion protein showed a positive CD peak at 230 nm, suggestive of some aromatic interaction between protease and VPg. Mutation of W43 in the VPg domain to phenylalanine/alanine abrogated the positive peak with concomitant loss in cis and trans proteolytic activities of the DN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggest a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in SeMV and probably by this mechanism the polyprotein processing could be regulated in planta. The three dimensional structure of the SeMV protease has revealed the residues involved in substrate binding (H298, T279, N308, R309), catalysis (H181, D216 and S284) and interaction with VPg (F269, W271, Y315, Y319). Mutational analysis of the residues forming the substrate binding pocket suggest that residues H298, T279 and R308 are absolutely required for the protease activity whereas N308 may not be so crucial. Further to delineate the interacting partners of W43, mutants of the above mentioned aromatic residues were generated. Results suggest that F269 and W271 residues of protease domain are involved in aromatic stacking interaction with VPg