Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome

Kaiming Zhang; Ivan N Zheludev; Rachel J Hagey; Raphael Haslecker; Yixuan J Hou; Rachael Kretsch; Grigore D Pintilie; Ramya Rangan; Wipapat Kladwang; Shanshan Li; Marie Teng-Pei Wu; Edward A Pham; Claire Bernardin-Souibgui; Ralph S Baric; Timothy P Sheahan; Victoria D'Souza; Jeffrey S Glenn; Wah Chiu; Rhiju Das

doi:10.1038/s41594-021-00653-y

Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome

Nat Struct Mol Biol. 2021 Sep;28(9):747-754. doi: 10.1038/s41594-021-00653-y. Epub 2021 Aug 23.

Authors

Kaiming Zhang^#^{1

2}, Ivan N Zheludev^#³, Rachel J Hagey^#⁴, Raphael Haslecker⁵, Yixuan J Hou⁶, Rachael Kretsch⁷, Grigore D Pintilie¹, Ramya Rangan⁷, Wipapat Kladwang³, Shanshan Li^{1

2}, Marie Teng-Pei Wu⁵, Edward A Pham⁴, Claire Bernardin-Souibgui⁴, Ralph S Baric^{6

8}, Timothy P Sheahan⁶, Victoria D'Souza⁵, Jeffrey S Glenn^{9

10}, Wah Chiu^{11

12

13}, Rhiju Das^{14

15

16}

Affiliations

¹ Departments of Bioengineering, James H. Clark Center, Stanford University, Stanford, CA, USA.
² MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China.
³ Department of Biochemistry Stanford University, Stanford, CA, USA.
⁴ Departments of Medicine (Division of Gastroenterology and Hepatology) and Microbiology & Immunology, Stanford School of Medicine, Stanford, CA, USA.
⁵ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
⁶ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁷ Biophysics Program, Stanford University, Stanford, CA, USA.
⁸ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁹ Departments of Medicine (Division of Gastroenterology and Hepatology) and Microbiology & Immunology, Stanford School of Medicine, Stanford, CA, USA. jeffrey.glenn@stanford.edu.
¹⁰ Palo Alto Veterans Administration, Palo Alto, CA, USA. jeffrey.glenn@stanford.edu.
¹¹ Departments of Bioengineering, James H. Clark Center, Stanford University, Stanford, CA, USA. wahc@stanford.edu.
¹² Biophysics Program, Stanford University, Stanford, CA, USA. wahc@stanford.edu.
¹³ CryoEM and Bioimaging Division, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA. wahc@stanford.edu.
¹⁴ Department of Biochemistry Stanford University, Stanford, CA, USA. rhiju@stanford.edu.
¹⁵ Biophysics Program, Stanford University, Stanford, CA, USA. rhiju@stanford.edu.
¹⁶ Department of Physics, Stanford University, Stanford, CA, USA. rhiju@stanford.edu.

^# Contributed equally.

Abstract

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

MeSH terms

A549 Cells
Animals
Base Sequence
COVID-19 / prevention & control*
COVID-19 / virology
Cell Line, Tumor
Chlorocebus aethiops
Cryoelectron Microscopy / methods*
Frameshift Mutation / genetics*
Genome, Viral / genetics
Humans
Models, Molecular
Nucleic Acid Conformation
Oligonucleotides, Antisense / genetics*
Oligonucleotides, Antisense / pharmacology
RNA, Viral / chemistry
RNA, Viral / genetics*
RNA, Viral / ultrastructure
Response Elements / genetics*
SARS-CoV-2 / genetics*
SARS-CoV-2 / physiology
SARS-CoV-2 / ultrastructure
Vero Cells
Virus Replication / drug effects
Virus Replication / genetics

Substances

Oligonucleotides, Antisense
RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding