GOF studies in MERS and

SARS countermeasures

Mark R. Denison M.D.

Vanderbilt University School of Medicine

Two natural, zoonotic, potentially pandemic

coronaviruses in 10 years:
Coronaviruses do not pause and do not deliberate
SARS-CoV: 32 countries >8000 cases in 2 months in 2003
High mortality in the elderly
High identity SARS-like CoV currently circulating in bats
No US Lab associated infections in >10 years of research
Already regulated by BSL3, Select Agent and DURC
No approved Vaccines or Therapeutics

MERS-CoV:

CURRENTLY active epidemic ongoing since 2012

>800 cases > 40% mortality (no reduction in mortality since 2013)
Mechanisms for ongoing outbreak unclear
No vaccines or therapeutics available
No small animal model available

Targets for Coronavirus Therapeutics

nsp3

nsp3
PL-protease

nsp5

nsp12

nsp14 nsp16

EM

An3

nsp5
protease

Protease Inhibitors (PL-pro, nsp5 (3CLpro)

Tested by purified proteins and in culture

Activity against one CoV does not predict activity against another CoV

Biochemical activity does not guarantee inhibition of infection in culture

or in vivo

Resistance arises rapidly: resistant mutants less fit than WT virus

No MERS-CoV small animal model available for testing

Goals of CoV therapeutics

Broadly active against diverse CoVs: MERS, SARS,

zoonotic precursor CoVs

Efficacy in vivo (small animals)

Slow or no resistance during therapeutic use

Known mechanism of activity

Progress at risk in current GOF definition and

pause for MERS-CoV

In vitro replication models

Adaptation for improved replication in culture

In vivo (mouse, small animal) models of replication and

pathogenesis

Genetic modification of mice

Passage adaptation for replication and pathogenesis

Passage for resistance, sequencing and reverse

genetic confirmation

Identify common mechanisms and determinants

Infidelity is Bad for

Coronavirus Relationships:
A Case Study

Would you take or give a live

vaccine with a virus that has
an engineered increased
mutation rate?
Why Not?

Should we engineer
mutator strains?

Coronaviruses proofread using a

3-to-5 exonuclease (ExoN)
nsp12

nsp14

RdRp

ExoN

EM

3
An

3-5 exonuclease (ExoN)

motif I
aa1

DE

motif II motif III

aa527

DE-D-D Exonuclease Superfamily Proofreading

Exonucleases in Bacteria and Eukaryotes
Mutations in DE-D-D motifs inactivate ExoN activity
(ExoN-)
(Snijder et al. 2003; Minskaia et al.,2006; Chen, et al., 2009)

Coronaviruses proofread using a

3-to-5 exonuclease (ExoN)
nsp12

nsp14

RdRp

ExoN

EM

3
An

3-5 exonuclease (ExoN)

motif I
aa1

DE

motif II motif III

aa527

AA

ExoN-

What is the effect of ExoN inactivation

(ExoN-) on virus replication fidelity?

CoV ExoN- mutants have a mutator

phenotype with a 20-fold increased
mutation rate

Substitutions
per genome
20.7x

SARS-CoV
ExoN

SARSExoN

Eckerle et al., J Virol 2007; PLoS

Pathogens 2010; Michelle Becker

Assumptions about RNA virus fitness

Generate populations with tremendous variation

Increased mutation rate = increased :

Adaptation and Fitness

Virulence
Public Health risk

SARS-ExoN- mutator strain is less fit

+
than wildtype SARS-ExoN
Input Ratio

Input Ratio

Input Ratio

1:1

10:1

100:1

Percent of total
genome RNA

100
80
60

SARS-ExoN

40

SARS-ExoN
20
0

1 2

3 4 5

Passage

1 2 3 4 5 1 2 3 4 5

Passage

Passage

Graham et al Nature Med 2012

SARS ExoN mutator virus is attenuated

in mouse model of lethal SARS

SARS-ExoN

Percent survival

100

102, 103, 104 PFU

80
2

10 PFU

60
40

20

10 PFU

10
PFU
0

SARS-ExoN

10

12

14

Time post infection (days)

Graham et al Nature Med 2012

ExoN- genotype and mutator phenotype

is stable in culture and in mice
Passage in culture >20X
Passage in aged mice
Persistent (60 day) infection in SCID mice
**non-redundant, non-complemented
**required for replication fidelity
Michelle Becker, Xiaotao Lu, Rachel Graham

Targeting nsp14-ExoN proofreading

for therapeutics
Wildtype CoVs are resistant to Ribavirin, RNA
mutagens, nucleoside analogs
ExoN- mutants are highly susceptible to acute
inhibition and lethal mutagenesis.
Inhibition of ExoN fidelity as combination Rx
with Ribavirin and developed nucleoside
analogs

Smith et al., PLoS Pathogens 2013

Our Assumptions: Are often wrong

Increased mutation rate does NOT favor virus fitness:
Fidelity is a target for vaccines and therapeutics.
Passage for adaptation and resistance in vitro and animal
models in vivo are essential components of therapeutic
development.
There are no bioinformatic or predictive safer alternative
approaches to identify unknown, unpredicted, and
unprecedented targets for countermeasures.
Limiting already safe and regulated research does not stop
coronavirus epidemics or adaptation of potential zoonotic
viruses to human disease.

Attenuation of Decreased Fidelity and

Increased Fidelity Mutants of RNA Viruses
CV-B3

polio

EV-71

FMDV

ChikV

SARS-CoV H5N1-Flu

Anti-mutator

Mutator
ExoN-

H5N1
influenza

Adapted from Smith Ann Rev Virol 2014

Acknowledgements
Vanderbilt
Clint Smith
Nicole Sexton
Michelle Becker
Xiaotao Lu

UNC
Ralph Baric
Rachel Graham
Amy Sims
Vineet Menachery

Elizabeth B. Lamb Center for

Pediatric Research
Craig-Weaver Chair in Pediatrics

R01 AI 108197
R01 AI26603
U19 AI109680