Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa

Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Kaiming Zhang, Ivan N. Zheludev, Rachel J. Hagey, Marie Teng-Pei Wu, Raphael

Haslecker, Yixuan J. Hou, Rachael Kretsch, Grigore D. Pintilie, Ramya Rangan, Wipapat

Kladwang, Shanshan Li, Edward A. Pham, Claire Bernardin-Souibgui, Ralph S. Baric,

Timothy P. Sheahan, Victoria D′Souza, Jeffrey S. Glenn, Wah Chiu, Rhiju Das

28/09/20

RefSeq: NC_045512.2, 2020; Rangan et al., RNA, 2020

~30,000nt = ~10MDa = encodes ~10,000aa

The genome is ‘positive sense’ = it looks like mRNA to the infected cell

This mRNA is split into “genes” called ‘Open Reading Frames’ (ORFs)

that tell ribosomes to make proteins

Often, these proteins are concatemers of many proteins,

‘Polyproteins’, which need to be cut up by viral proteases

So, while the genome encodes ~12 ORFs,

it produces many tens of unique proteins

SARS-CoV-2 has a huge ssRNA genome

Most of the SARS-CoV-2 genome encodes 1 special ORF

ORF1ab

de Wit et al., Nat. Rev. Microbio., 2016

ORF1ab has one

translation start site

(arrow) but makes two

polyproteins:

PP1a: 4405aa

PP1ab: 7096aa

All ribosomes start here for ORF1ab

PP1a: early viral proteins

PP1ab: early viral proteins + late viral proteins

The ratio of PP1a to PP1ab made is tightly controlled (and vital to viral fitness)

de Wit et al., Nat. Rev. Microbio., 2016

All ribosomes start here for ORF1ab

Zoom in

The ratio of PP1a to PP1ab made is tightly controlled (and vital to viral fitness)

de Wit et al., Nat. Rev. Microbio., 2016

All ribosomes start here for ORF1ab

Zoom in

Ribosome translating the ORF1a part

of ORF1ab

The ratio of PP1a to PP1ab made is tightly controlled (and vital to viral fitness)

de Wit et al., Nat. Rev. Microbio., 2016

All ribosomes start here for ORF1ab

Zoom in

Usually, the ribosome reaches the STOP at 13481 and finishes, making PP1a

PP1a

The ratio of PP1a to PP1ab made is tightly controlled (and vital to viral fitness)

de Wit et al., Nat. Rev. Microbio., 2016

All ribosomes start here for ORF1ab

Zoom in

Occasionally, the ribosome changes reading frame, avoiding the STOP

and continues to add ~3000aa to make PP1ab

STOP

avoided

The ratio of PP1a to PP1ab made is tightly controlled (and vital to viral fitness)

de Wit et al., Nat. Rev. Microbio., 2016

All ribosomes start here for ORF1ab

Zoom in

This ‘frameshifting’ decision is mediated

by RNA (genome) structure

Frameshift Stimulation Element

Fernandes, et al., bioRxiv, 2020; Ahn, et al., Antiviral Research, 2011; Park, et al., JACS, 2011; Kelly, et al., J. Biol. Chem, 2020; Neupane, et al. bioRxiv, 2020

Ph

ylo

P S

co

re

Conserved possible restricted escape

The FSE is an attractive drug target

and validated in CoV-2Leads have been made against SARS-CoV

asPNAviral replication

small moleculeIn vivo dual luciferase

Cryo-EM allows for rapid naked RNA structure determination

Kappel et al., Nat. Met. - 2020

Ribosolve:

~1 week

the preprint

Figure 4 – the pseudoknot does not form a knot, but it might make a quasi-knot

Apologies for low resolution

Figure 2 – cryo-EM and models

Figure 2 – cryo-EM and models

Kappel et al., Nat. Met. - 2018

estimated accuracy = 5.9Å

Figure 2 – cryo-EM and models

‘literature’

SHAPE-CE

M2seq

Figure 2 – cryo-EM and models

Figure 2 – cryo-EM and models

Where are we?

Kappel et al., Nat. Met. - 2020

Could we insert a known structure in a known location?

Figure 3 – nanostructure tagging for cryo-EM cross-validation

?

??

Figure 3 – nanostructure tagging for cryo-EM cross-validation

Figure 3 – nanostructure tagging for cryo-EM cross-validation

Figure 3 – nanostructure tagging for cryo-EM cross-validation

Figure 4 – the pseudoknot does not form a knot, but it might make a quasi-knot

Rangan et al., bioRxiv, - 2020b; Omar et al., bioRxiv, - 2020

Figure 4 – the pseudoknot does not form a knot, but it might make a quasi-knot

Figure 4 – the pseudoknot does not form a knot, but it might make a quasi-knot

Jose Gustavo Chacon

Extra Figure 4 – but what about the alternative Stem 1?

Extra Figure 4 – but what about the alternative Stem 1?

Extra Figure 4 – so Stem 2 has to ‘latch’ to allow threading

Secret Figure 4 – so Stem 2 has to ‘latch’ to allow threading – maybe it can?

Figure 4 – the FSE might have conserved druggable sites

Acknowledgements

Chiu lab: • Kaiming Zhang• Grigore D. Pintilie• Shanshan Li• Wah Chiu

Das lab:• Rachael Kretsch• Ramya Rangan• Ann Kladwang• Eesha Sharma• Rhiju Das

Glenn lab:• Rachel Hagey• Claire Solange Audrey

Bernardin• Menashe Elazar• Ed Pham• Jeff Glenn

D’Souza lab:• Raphael Haslecker• Marie Teng-Pei Wu• Victoria D’Souza

Stanford SGF:• Paul Berg

Stanford:• COVID continuation team• Jessica Corkern• PAN facility