Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly

Abstract

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N∆65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

All-atom normal mode dynamics of HIV-1 capsid

“…We use four capsids as test cases to evaluate the quality of BOSE modes. The four capsids are: capsid of Satellite Tobacco Necrosis Virus (STNV, pdb-id: 4V4M) [ 31 ], capsid of Sesbania mosaic virus (SeMV, pdb-id: 4Y5Z) [ 32 ], a mutant structure of the capsid of Grouper nervous necrosis virus (GNNV, pdb-id: 4RFT) [ 33 ], and capsid of a lumazine synthase from the thermophilic bacterium Aquifex aeolicus (AaLS, pdb-id: 5MPP) [ 34 ]. The four capsids all have icosahedral symmetry.…”

Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein

“…No special features such as pseudo-translation or twinning were observed in the data set. Examination of the unit-cell parameters, which were typical of a small protein crystal, suggested that the chimeric protein had not assembled into virus-like particles, as observed in other similar experiments (Gulati et al, 2016). Because the unit-cell parameters were different from those of the other crystals obtained for various constructs of the SeMV coat protein, it was speculated that a proteolytic cleavage product of the viral coat protein might have been crystallized.…”

“…In one of the crystallization trials, a chimeric construct of the well studied Sesbania mosaic virus (SeMV; Subramanya et al, 1993;Murthy et al, 1997) in which the N-terminal 65 residues were replaced by Staphylococcus aureus protein A (SpA) (NÁ65 CP-B; Gulati et al, 2016) appeared to crystallize in space group P2 1 using the hanging-drop vapour-diffusion method. The crystallization drop consisted of 2 ml protein sample (20 mg ml À1 ) and 2 ml crystallization buffer (0.1 M bistris pH 6.5, 20% PEG MME 5000) incubated at 22 C. Crystals were obtained in about two weeks.…”

“…Unfortunately, the protein could not be recrystallized in the same form to carry out experimental phasing. The crystals obtained in subsequent trials belonged to space groups P1 or C2 and their structures have been reported (Gulati et al, 2016). This was a puzzling result as the chimeric protein is identical in sequence to the viral coat protein except for the inserted polypeptide, and hence the polypeptide structure of NÁ65 CP should have provided an adequate phasing model.…”

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

“…The resultant plasmid (pRSETC CP sdm) was digested with AfeI enzyme. B domain gene, PCR amplified using pRSETC NΔ65B CP 45 as template and B domain specific primers ( Table 1 ), was ligated to AfeI digested pRSETC CP sdm to form pRSETC SLB His. For removal of the N terminal Histidine tag, the entire construct was PCR amplified using CP specific primers ( Table 1 ) digested with EcoRI and inserted in NdeI end filled and EcoRI cut pRSETC vector.…”