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