Molecular Mechanism of Folding of Nsp12 and Assembly of the SARS-CoV-2 RNA Polymerase Complex by the Cytosolic Chaperonin CCT - PROJECT SUMMARY
The COVID-19 pandemic created the greatest infectious threat to global health in 100 years,
and monumental efforts have been made by the scientific community to combat the SARS-CoV-
2 virus. This proposal seeks to extend this effort by investigating a mechanism by which SARS-
CoV-2 hijacks the host cell chaperone system to replicate itself. We have evidence that the
SARS-CoV-2 RNA polymerase (RdRp) co-opts the cytosolic chaperonin containing TCP-1
(CCT, also called TRiC) to assemble the active polymerase complex. CCT is a large (1 MDa)
protein-folding machine that plays a major role in the cellular chaperone network responsible for
maintaining the proteome in good working condition. It uses ATP hydrolysis-driven
conformational changes to assist cytosolic proteins with multiple domains, complex folding
trajectories, or obligate binding partners to achieve their native state and assemble into
complexes. In addition to folding cellular proteins, CCT has been shown to bind several viral
proteins and contribute to viral replication of HIV, hepatitis C, influenza A, rabies, Zika and
reovirus. These observations show that CCT is a common host chaperone used by diverse
viruses to fold viral proteins, assemble viral complexes, and support viral replication. Based on
these findings, we initiated an investigation of the role of CCT in SARS-CoV-2 replication. Here,
we present robust preliminary evidence indicating that the SARS-CoV-2 non-structural protein
12 (Nsp12), the catalytic subunit of the RNA polymerase, is folded by CCT and that CCT
contributes to RdRp complex formation and SARS-CoV-2 replication. In Aim 1, we propose to
thoroughly test this hypothesis using multiple experimental approaches. In Aim 2, we propose to
determine high-resolution structures of the complex between Nsp12 and CCT. We have isolated
an Nsp12 folding intermediate bound to CCT and have determined preliminary structures of the
complex by cryogenic electron microscopy (cryo-EM). Further cryo-EM analysis will yield a high-
resolution structure of the Nsp12-CCT complex, which will be invaluable in guiding the design of
therapeutics to block Nsp12 folding by CCT, inhibit formation of the RdRp complex, and disrupt
viral replication.
