NSRRC Activity Report 2023

052 NSRRC ACTIVITY REPORT 2023 lead to pH-dependent particle size, the size fluctuations of T = 4 LSV2 VLP were examined using X-ray crystallography and small-angle X-ray scattering (SAXS). In the X-ray crystallographic analysis, the diameter of the T = 4 LSV2 VLP, measured to be 450 Å based on crystal packing, was marginally smaller than the size range identified by cryo- EM ( Fig. 2(b) ). During SAXS analysis, the pH conditions of LSV2 VLP were directly adjusted from pH 7.5 to pH 6.5 and 8.5. Conversely, the particle sizes of T = 4 and T = 3 LSV2 VLPs align closely with both the expanded and unexpanded conformations observed in the cryo- EM analyses ( Fig. 2(c) ). Interestingly, in many cryo-EM images of LSV2 VLP, linear structures comprising several trapezoid-shaped capsomeres arranged in a domino-like architecture were observed. This structural foundation of trapezoid-shaped capsomeres and the configuration resembling a domino scaffold offers insights into the mechanism of capsid assembly ( Fig. 2(d) ). Finally, mass spectrometry was used to characterize the autoproteolysis behavior of CP in a mutagenesis study, mimicking the cleavage site. In summary, the atomic-resolution structures of LSV2 and delta-N48 LSV1 VLPs not only offer significant structural insights into viral capsids but also enable a detailed examination of the RNA-binding site and the driving forces behind capsid assembly. Despite the evolutionary and biological divergence of LSV, which has been classified into at least eight subgroups, many gene sequences of CP are conserved among strains of LSV. This study presents a structural model applicable to all strains of LSV, suggesting their self-assembly into common T = 4 tetravirus structures with single CP subunits and their ability to infect host cells through a similar entry pathway. (Reported by Nai-Chi Chen) This report features the work of Chun-Jung Chen and his collaborators published in Nat. Commun. 14 , 545 (2023). TPS 05A Protein Microcrystallography SP 44XU Macromolecular Assemblies (International Collaboration) • Protein Crystallography • Biological Macromolecules, Protein Structures, Life Science TPS 13A Biological Small-angle X-ray Scattering • BioSAXS • Structural Transitions of Macromolecules in Solution References 1. D. Goulson, E. Nicholls, C. Botías, E. L. Rotheray, Sci- ence 347 , 1255957 (2015). 2. D. L. Cox-Foster, S. Conlan, E. C. Holmes, G. Palacios, J. D. Evans, N. A. Moran, P.-L. Quan, T. Briese, M. Hornig, D. M. Geiser, V. Martinson, D. vanEngelsdorp, A. L. Kalkstein, A. Drysdale, J. Hui, J. Zhai, L. Cui, S. K. Hutchison, J. F. Simons, M. Egholm J. S., Pettis, W. I. Lipkin, Science 318 , 283 (2007). 3. N. C. Chen, C. H. Wang, M. Yoshimura, Y. Q. Yeh, H. H. Guan, P. Chuankhayan, C. C. Lin, P. J. Lin, Y. C. Huang, S. Wakatsuki, M. C. Ho, C. J. Chen, Nat. Commun. 14 , 545 (2023). Fig. 2 : (a) The cryo-EM maps illustrate T = 4 (left) and T = 3 (right) LSV2 VLPs with diameters of 482 and 438 Å, respectively, under acidic pH conditions, represented in dark khaki. Meanwhile, VLPs with respective diameters of 492 and 448 Å under neutral pH conditions are depicted in transparent rendering. (b) Molecular packing of the T  =  4 LSV2 VLP in crystals is arranged in a unit cell with the space group P 4 2 . (c) The intensity vs. q profile of LSV2 VLPs as obtained from SAXS in different buffer solutions, with the buffer contribution subtracted. The T =4 and T =3 LSV2 VLPs, each with distinct particle sizes, are depicted in black and blue, respectively, under pH conditions of 8.5 and 6.5. (d) Schematic diagrams illustrating the presumed self-assembly mechanism of both T = 4 and T = 3 complete capsids of LSVs, highlighting the impact of pH conditions on the dynamic motion of VLP particles. The homo-trimeric capsomeres constitute a fundamental structural unit during the assembly process. [Reproduced from Ref. 3]