Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses

David Chmielewski; Guan-Chin Su; Jason T Kaelber; Grigore D Pintilie; Muyuan Chen; Jing Jin; Albert J Auguste; Wah Chiu

doi:10.1093/pnasnexus/pgae102

Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses

PNAS Nexus. 2024 Mar 7;3(3):pgae102. doi: 10.1093/pnasnexus/pgae102. eCollection 2024 Mar.

Authors

David Chmielewski¹, Guan-Chin Su^{2

3}, Jason T Kaelber⁴, Grigore D Pintilie^{2

3}, Muyuan Chen⁵, Jing Jin^{6

7}, Albert J Auguste^{8

9}, Wah Chiu^{1

2

3

5}

Affiliations

¹ Biophysics Graduate Program, Stanford University, Stanford, CA 94305, USA.
² Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
³ Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.
⁴ Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
⁵ Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025, USA.
⁶ Vitalant Research Institute, San Francisco, CA 94118, USA.
⁷ Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA.
⁸ Department of Entomology, College of Agriculture and Life Sciences, Fralin Life Science Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
⁹ Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.

Abstract

Alphaviruses are spherical, enveloped RNA viruses with two-layered icosahedral architecture. The structures of many alphaviruses have been studied using cryogenic electron microscopy (cryo-EM) reconstructions, which impose icosahedral symmetry on the viral particles. Using cryogenic electron tomography (cryo-ET), we revealed a polarized symmetry defect in the icosahedral lattice of Chikungunya virus (CHIKV) in situ, similar to the late budding particles, suggesting the inherent imperfect symmetry originates from the final pinch-off of assembled virions. We further demonstrated this imperfect symmetry is also present in in vitro purified CHIKV and Mayaro virus, another arthritogenic alphavirus. We employed a subparticle-based single-particle analysis protocol to circumvent the icosahedral imperfection and boosted the resolution of the structure of the CHIKV to ∼3 Å resolution, which revealed detailed molecular interactions between glycoprotein E1-E2 heterodimers in the transmembrane region and multiple lipid-like pocket factors located in a highly conserved hydrophobic pocket. This complementary use of in situ cryo-ET and single-particle cryo-EM approaches provides a more precise structural description of near-icosahedral viruses and valuable insights to guide the development of structure-based antiviral therapies against alphaviruses.

Keywords: alphavirus; cryo-EM; cryo-ET; imperfect icosahedral symmetry.

Abstract

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