Folding of the SARS‐CoV‐2 RNA Polymerase by the Cytosolic Chaperonin CCT

Abstract

The ongoing Covid‐19 pandemic, caused by the highly transmissible SARS‐CoV‐2 virus, constitutes the worst global public health crisis of the past century. Although effective vaccines have been developed, administration has been slow, new variants continue to emerge, and there remains a lack of effective antivirals to treat severe cases. Thus, there remains a significant need to understand the mechanisms of SARS‐CoV‐2 infection and replication to identify new potential therapeutic targets. To this end, we investigated whether SARS‐CoV‐2 might depend on the host cell chaperone system, particularly the essential cytosolic chaperonin, CCT, to fold or assemble any of its proteins. CCT has previously been shown to be required for the replication of several other viruses including reoviruses and zika virus. We screened likely candidates among the SARS‐CoV‐2 proteins for co‐immunoprecipitation with CCT and identified an interaction with the RNA‐dependent RNA polymerase, Nsp12. To confirm Nsp12 was a substrate, we depleted cells of CCT, then transfected with Nsp12 and observed a 40% decrease in Nsp12 expression in CCT‐depleted cells compared to the control. This decrease is consistent with what we have observed for other known CCT substrates. Additionally, CCT‐depleted cells infected with live SARS‐CoV‐2 produced a 50% decrease in viral titer compared to controls, indicating that CCT is important for viral replication. A preliminary cryo‐EM structure of the Nsp12‐CCT complex shows a very large mass identifiable as Nsp12 inside of CCT that extends from the equatorial domains up and out through one of the two folding cavities. The structure stands to both reveal an important step in SARS‐CoV‐2 replication and to answer a long‐standing question regarding how CCT can accommodate very large substrates.