Architecture of a SARS-CoV-2 mini replication and transcription complex

Abstract

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.

Fig. 1. Architecture of mini RTC.

a The scaffold of RNA used in the structural study is shown. b Domain organization of each component in mini RTC. The color scheme for nsp7, nsp8, and nsp12 are generally the same as those used in our previous work with slight modifications. Domains of nsp13 are shown in different colors with labels. c Helicase activities. The helicase activities of mini RTC, the individual nsp13 (nsp13), mini RTC with an R365A mutation in nsp12, and mini RTC with a T216A mutation in nsp13. The velocities of helicase activity are shown as the mean of two independent experiments. d Cryo-EM density of mini RTC (represented by form 1) is shown in three perpendicular views. The color scheme is the same as used in (b).

Fig. 2. Binding of 5′ extension of RNA template in nsp13-2.

a Cutaway view of RNA primer–template pair bound to mini RTC. The structures of nsp7, nsp8, nsp12, and nsp13 are covered by a molecular surface with the same color scheme in Fig. 1. The bound RNA primer–template pair is exhibited as colored cartoons. The missing nucleotides are denoted as a dashed line. b Key interactions nsp13-2 and 5′ extension of RNA template. The molecule of nsp13-2 is shown as a cartoon diagram, and key residues involved in the binding with RNA are shown as colored sticks. The dashed lines indicated the bonds with a distance of less than 3.5 Å.

Fig. 3. Two orientations of nsp13-1.

a Structural comparison of two mini RTCs in two perpendicular views. The structural parts with high similarity in two mini RTCs, including nsp7–nsp8–nsp12-RNA and nsp13-2, are covered by a molecular surface with the same color scheme in Fig. 1. Nsp13-1 molecules in form 1 and form 2 mini RTC are displayed as white and red cartoons. b The interaction regions of nsp13 molecules with other components of mini RTC or with each other. The structural details for region 1 (c, d), region 2 (e, f) and region 3 (g, h) are enlarged. Form 1 is in c, e, g; form 2 is in d, f, h. Dashed lines denoted the interactions with a bond distance of less than 3.5 Å.

Fig. 4. A proposed model for the helicase-polymerase coupling in the formation of SARS-CoV-2 RTC.

Nsps composed mini RTC and template-primer RNAs are shown as schematic diagrams. Step 1, nsp12 bound nsp7, and nsp8 to constitute central RTC, waiting for a template RNA unwinding. The nsp13 molecule keeps in an inactive state. Step 2, an nsp13 (nsp13-1) contacts central RTC to form a platform to recruit the nsp13-2. Step 3, nsp13-2 is assembled into mini RTC, and contacts with nsp13-1, allowing the bound RNA translocating towards nsp12 activity center. Step 4, the unwinded single-strand RNA template passes through the RNA-binding channel of nsp13-2 and extends to nsp12 active center for the subsequent nascent RNA synthesis.