of 53
1
Variant mutation in SARS
-CoV-
2 nucleocapsid enhances viral infection via altered
genomic encapsidation
Hannah C. Kubinski
1,#
, Hannah W. Despres
1,#
, Bryan A. Johnson
2,3,4
, Madaline
M. Schmidt
1,&,
,
Sara A.
Jaffrani
1
, Margaret G. M
ills
5
, Kumari
Lokugamage
2
, Caroline
M. Dumas
6
, David J.
Shirley
7
, Leah K. Estes
2
, Andrew
Pekosz
8
, Jessica
W. Crothers
9
,
Pavitra Roychoudhury
5
,
Alexander L. Greninger
5, 10
, Keith R. Jerome
5, 10
, Bruno Martorelli
Di Genova
1
, David H. Walker
11
,
Bryan A. Ballif
6
, Mark S. Ladinsky
12
, Pamela J. Bjorkman
12
, Vineet
D. Menach
ery
2,13,14
, Emily A.
Bruce
1,
*
1
Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine,
University of Vermont, Burlington VT, 05405, USA.
2
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston,
Texas,
USA
3
Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX,
USA
4
Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX, USA
5
Virology Division, Department of Laboratory Medicine and Pathology, University of Washington,
Seattle WA 98195, USA.
6
Department of Biology, University of Vermont 109 Carrigan Drive, 120A Marsh Life Sciences,
Burlington VT 05404, USA
7
Faraday, Inc. Data Science Department. Burlington VT, 05405, USA.
8
W. Harry Feinstone
Department of Molecular Microbiology and Immunology, The Johns
Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
.
9
Department of Pathology and Laboratory Medicine, Robert Larner, MD College of Medicine,
University of Vermont, Burlington, VT, USA.
10
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle
WA 98109, USA.
11
Department of Pathology
, University of Texas Medical Branch, Galveston, Texas,
USA
.
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2
12
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena,
CA. 91125, USA
1
3
World Reference Center of Emerging Viruses
and Arboviruses, University of Texas Medical
Branch, Galveston, Texas,
USA
.
1
4
Center
for Biodefense and Emerging Infectious Diseases, University of Texas Medical
Branch, Galveston, Texas,
USA
.
&
Current address: Microbiology and Molecular
Genetics Graduate Program, Emory University,
Atlanta, Georgia, USA.
# H
CK
and
HWD
contributed equally to this work.
*Correspondence:
Emily.bruce@med.uvm.edu
(EAB)
ABSTRACT
The
evolution of SARS
-
CoV
-
2
variants and their respective phenotypes
represent
s
an
important
set of tools to understand
basic
c
oronavirus biology as well as the public health
implications of individual mutations
in variants of
concern.
While mutations outside of Spike are
not well studied, the
entire
viral genome
is undergoing
evolutionary selection
, particularly the
central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation
(G215C)
,
characteristic of the Delta variant
,
that introduces a novel cysteine into this linker
domain
,
which results in the formation of a disulfide bon
d and a stable N
-
N dimer.
Using reverse
genetics, we
determined
that this cysteine residue
is necessary and sufficient for stable dimer
formation in a W
A1
SARS
-
CoV
-
2 background
, where it results in significantly increased viral
growth both
in vitro
and
in vivo
.
Finally, we demonstrate that the N:G215C virus packages more
nucleocapsid per virion and that individual virions
are larger, with elongated morphologies.
INTRODUCTION
The coronavirus disease of 2019 (COVID
-
19)
pandemic originated from the emergence
of
S
evere
A
cute
R
espiratory
S
yndrome
C
oronavirus 2 (SARS
-
CoV
-
2
)
in late 2019
1
.
Subsequent
.
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3
worldwide spread and sustained transmission over the past four years
, combined with near real
-
time access to
viral genomic surveillance data
, has revealed a detailed picture of
SARS
-
CoV
-
2
evolution in the human population.
In
dividual mutations that conferred an evolutionary
advantage were quickly selected and maintained in viral lineages, leading to the emergence of
novel
V
ariants
of
C
oncern distinguished by increased immune evasion,
transmission,
disease
burden or infectivity.
U
nderstanding the
biological function
of
the
individual mutations
that led to
the emergence of novel variants of concern has
important implications for
public health
consequences
as well as
our fundamental understanding of basic
coronavirus biology
.
Most studies
concerning these mutations have focused on genetic changes within the
spike (S) protein, due
to concerns
that sufficiently novel spike proteins can
allow viral
escape
from
the immune memory induced by vaccination or prior infection
2
5
.
However,
mutations
elsewhere in the viral genome can
play
key
role
s
in viral
replication and
pathogenesis
6
8
.
The
nucleocapsid
(N)
protein
in particular
is a genetic ‘hotspot’ for
mutations
across variants
,
with
several mutations
with
in
the
flexible
linker region
associated with increased replication and
pathogenesis
8
. Given the many attributed roles of N, including RNA encapsidation
9
,
production
of viral RNA
10
13
, and
viral assembly
through interactions with M
14
16
, mutations within this
protein
are poised to have large impacts on the viral lifecycle.
Additionally,
N is a highly
conserved protein
among coronaviruses, making
th
e N protein
a versatile diagnostic marker and
potential vaccine target candidate
17
.
We have identified a novel cysteine within the linker region
of N
that was selected
and
maintained within the
SARS
-
CoV
-
2
D
elta
variant.
This introduction of a cysteine residue
within
the nucleocapsid is unique amongst pandemic
-
causing betacoronaviruses and has major
structural implications as it
results in the production of a stable N
-
N dimer linked by a disulfide
bond.
Here we describe the impact of
this
novel cysteine with
in the
N protein and the
role it
plays in viral replication and
particle formation
.
We demonstrate that this cysteine
promote
d
N
oligomerization
,
and that
the
D
elta mutation (G215C)
result
ed
in substantially increase
d
viral
.
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replication kinetics in primary
differentiated human bronchial cells
.
The N:G215C mutation also
increased viral replication in the nasal washes and lungs of infected Syrian golden hamsters,
while paradoxically delaying weight loss.
Finally, t
he N:G215C virus
package
d
substantially
more N
per virion
and
many of the virions display
ed
elongated
morphology.
Together, our data
suggest that the
D
elta N:G215C mutation
increases levels of nucleocapsid oligomerization
which drives increased
packaging of N in
to
mature virions and results in significant increases in
viral replication both
in vitro
and
in vivo
.
RESULTS
Introduction of
a novel cysteine
in
the
nucleocapsid
linker region of
Variants of Concern
The unprecedented access to
sequences of SARS
-
CoV
-
2 genomes acquired from
individual infected people in near real
-
time throughout the COVID
-
19 pandemic
has revealed a
detailed picture
of
the
evolution
of this viral pandemic
over the last
four years.
Mutations
introduced by the viral RNA
-
dependent RNA
-
polymerase
led to the emergence of distinct
lineages, characterized by suites of different mutations. Variants
that met certain public health
benchmarks, termed Variants of Concern (VOCs)
,
contained individual mutations in viral
proteins that
conferred distinct evolutionary advantages.
These properties included the ability to
evade prior immunity
, increased transmission and altered virulenc
e
.
The
D
elta variant, which
emerged in mid
-
2021
possessed all the properties above, and contained
a series of novel
mutations,
including key changes
in the spike (S) protein
7
.
While the function of the mutations in
s
pike ha
s
been
intensively studied
4,18,19
,
the
majority of Delta sub
-
lineages also contained a
unique mutation in the nucleocapsid (N) protein
which
convert
ed
a glycine
into a cysteine at
position 215 (G215C).
This point
-
mutation
dominated
the Delta
lineages
(Fig
.
1
A
)
and
bioinformatic
analysis suggested that its evolutionary advantage was distinct from co
-
occur
r
ing
mutations in spike
or nucleocapsid
20
.
.
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The
G215C mutation sits in the disordered linker region of the nucleocapsid protein,
which lies
between a N terminal RNA
-
binding domain (RBD) and a C terminal dimerization
domain
(Fig
1B
). Th
e
introduction
of
a cysteine in the SARS
-
CoV
-
2 nucleocapsid is unique
amongst
zoonotic
Betacoronavirus
, as
neither
SARS
-
CoV
,
MERS
or
other
SARS
-
CoV
-
2
variants’
nucleocapsids
contained a
ny
cysteine
s
(Fig
1C
). Furthermore
, while other
c
oronavirus
nucleocapsid proteins do
contain cysteines, they are largely absent from the linker region
(
Fig
S1).
Surprisingly, when analyzing the
sequences of our panel of VOCs obtained from clinical
specimens
, we discovered that two other
variant
(
B
eta &
I
ota)
isolates also
contained a cysteine
within the N protein
(Fig
1C
)
.
Interestingly, all three of these mutations sit within
the
intrinsically
disordered
linker
region of N
(also termed
N3/sN3
)
between N2 (RNA binding domain) and N4
(dimerization domain).
Both our
B
eta (
B
.1.351
)
and
I
ota (
B.1.526
)
stocks
contained
novel
cysteine
s in
the linker region
,
R
185C
(99.7% of reads)
and G234C
(100% of reads)
,
respectively
(Fig
1C
)
.
Since
the introduction of a cysteine residue
would allow for the formation
of a
new
disulfide
-
bonded N
-
N
dimer
complex
, we predicted that this mutation could have major
impacts on the
secondary, tertiary and
/or
quaternary protein structure of the nucleocapsid
protein.
C
ysteine
in the N linker promotes
the formation of a stable
N
-
N
dimer
To
test
if
these
novel
cysteines would make a
more stable
N
-
N dimer, we
visualized
the
N
from our panel of variant isolates
by
western blot
under non
-
reducing conditions
. We infected
VeroE6
-
TMPRSS2 cells with
wildtype virus (ancestral SARS
-
CoV
-
2 from the WA1 infectious
clone) as well as
low passage stocks of
the
A
lpha,
B
eta,
G
amma,
D
elta,
E
psilon,
I
ota,
M
u, and
O
micron variants isolated from clinical samples.
A
ll viruses produced a band of the expected
molecular weight
(~
47
kD
a
)
for the N monomer, and the majority also showed a series of
truncation products
(indicated with <)
that
we hypothesize to be caspase cleavage
products
21
(Fig
2
A
)
.
As predicted
, t
he three variants which contained the novel cysteine residue
(Beta,
.
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Delta, Iota) produced
a second band detected at twice the molecular weight of the expected N
monomer
(
Fig
2
A
, see *
)
.
Of the three variants, Delta
(G215C)
produced the greatest level of
dimerized
-
N,
with
Iota (G243C) and
Beta (
R185C) each producing slightly lesser
amounts (Fig
2
B).
While it
is known that
coronavirus N protein
s
typical
ly
form dimers via
their
dimerization
domain
s
,
this is mediated by
non
-
covalent bonds and the canonical N
-
dimer was not seen
by
SDS
-
PAGE
gel/western blot
for viruses that lacked cysteines (WT, Alpha, Gamma, Epsilon, Mu
and Omicron).
To further test whether this higher molecular weight band represented a disulfide
-
bonded form of N
-
N dimer we performed several additional experiments
. First,
we prepared
samples
under strong reducing conditions
(10
mM DTT)
, where we observed
the
higher
molecular weight band
was
eliminated
(
Fig
S
2
A
)
. To
ensure that
this disulfide bond truly
occurred within infected cells and was not a post
-
lysis
artifact,
lysates were
harvested
in the
presence of
n
-
ethylmaleimide
(NEM)
. NEM binds irreversibly to free cysteines and prevents the
formation of post
-
lysis disulfide bonds,
and the band belonging to the putative N dimer remained
visible in these
conditions
(
Fig
S
2
B
).
To
confirm the putative dimer was not an artifact of the
monoclonal antibody used, we probed lysates with a second independent antibody
(Fig S2C/D)
.
Slight differences in cleavage products were seen with the two antibodies (see *), confirming
that
the antibodies recognized different epitopes, but
the
higher
molecular
weight band
was
visible in both
conditions
(
Fig
S
2
C
-
E
).
To
ensure that the higher molecular band did not
represent a disulfide bond formed between N and a cellular
or viral
protein of
equivalent
weight,
we
immun
o
precipitated N from cells
infected with WT or Delta SARS
-
CoV
-
2
and performed
mass
spectrometry
on tryptic peptides from
gel slices cut from the
regions corresponding to the
monomer and
putative ‘
dimer
(Fig S
2
F
).
In the Delta infected sample,
most
peptides detected in
the
‘dimer’
gel slice correspond
ed
to
N, and roughly half the
total N peptides were detected in
the ‘dimer’ vs ‘monomer’ slice (
Fig S
2
G
).
Furthermore, there were no cellular peptides of similar
abundance found in the ‘dimer’ slice, with the most abundant cellular protein found at >1/5
th
the
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levels of N
(Table S1).
Given the structural location of these cysteines in the linker region, and
the fact that N is known to oligomerize into higher order structures
22,23
, we think it is likely that
the introduction of this disulfide bond promotes the formation of a tetramer or higher order
structure by stabilizing the bond between two non
-
covalently
-
bonded dimers.
N dimer stability dependent on novel cysteines in linker region.
As the production of a disulfide bond in the reducing environment of the cytoplasm is
unusual, we
next determine
d
whether the formation of this stable N
-
N dimer required the
context of infection
.
SARS
-
CoV
-
2 replication produces many membran
ous
compartments with
limited cytoplasmic access
that could shield N during authentic infection
.
W
e created plasmid
expression constructs of the WT, Delta, Beta and Iota nucleocapsid sequences and transfected
these into HEK
-
293T cells
, in conjunction with constructs where each cysteine was mutated
back to the
canonical residue in the WT sequence (Delta C215G, Beta C185R, Iota C243G).
While all three variant constructs (Delta, Beta, Iota) were capable of
producing
stably dimerized
N (Fig
3
A),
Delta produced this
product to the highest levels while the Beta construct did not
consistently
form
visible
,
stable
dimer (Fig
3
B).
In each case, w
hen the cysteine
wa
s reverted to
its original amino acid the
stabilized
dimer
wa
s not made
(Fig
3
A/B
)
.
Th
ese
data sugges
t
that, at
least in the case of Delta and Iota, the presence of other viral proteins/RNA
and the formation of
double membrane vesicles (DMVs)
are
not required for stably dimerized N formation
,
and the
presence
of a cysteine at 215/243
in the Delta/Iota backgrounds
is sufficient
.
Impact of the N:G215C mutation on viral growth
in vitro
and
in vivo
As
bioinformatic evidence suggests
th
at Delta variants containing the
G215C mutation
outcompeted those that contained the ancestral glycine at
that position
20
we wanted to examine
the impact of the G215C mutation on viral growth kinetics.
Since
WT
SARS
-
CoV
-
2 (WA1
/2020
)
does not produce the
stably
dimer
ized N
in infection (Fig
2
A
), we
investigated if the introduction
.
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of a cysteine
at 185, 215 or 243 (B
eta,
Delta, I
ota
) was sufficient to mediate stable dimer
formation in a WT background
. Using plasmid constructs, we transfected HEK
-
293T cells with
either
WT
N
,
or
WT
N containing a single point mutation (
R
185C, G215C, and G234C). When
visualized via western blot in non
-
reducing conditions, only the construct which contained the
D
elta G215C mutation could produce the
stabilized
dimer
, suggesting that this mutation is both
necessary and sufficient for stable dimer formation
(
Fig
3C,D
).
As
inserting the G215C mutation in the WT background was sufficient to confer
dimer
formation, we used a well
-
established reverse genetics system
24
to
rescue
an infectious clone
containing the G215C nucleocapsid mutation
(N: G215C)
in the SARS
-
CoV
-
2 WA1 backbone
(Fig 4A)
. We used
a
neon
-
green reporter
virus in the WA1
background (
mNG SARS
-
CoV
-
2
)
,
which is
genetically identical to WA1 except that ORF7 has been replaced with a neon green
fluorescent reporter).
Notably,
mNG SARS
-
CoV
-
2
is attenuated compared to
the parental
WA1
,
due to the
replacement of ORF7a with the mNG reporter
8
.
As expected, the N:G215C virus
produced the stable N dimer
in infected Vero
-
TMPRSS2 cells under non
-
reducing
conditions
(Fig
4
B).
In a
multi
-
cycle growth curve
, the WT and N:G215C viruses grew to
identical peak
titers, with very similar growth kinetics. We did not
e that at the earliest timepoint (7hpi), the WT
virus shows an ~1 log drop in viral titer (representing the loss in infectivity of
the original
inoculum),
while
the N: G215C virus
did not display such a drop
(Fig
4
C)
, though
the biological
significance (if any) of this observation is unclear
.
Next
, as
VeroE6 cell
s are not a physiologically relevant target for SARS
-
CoV
-
2 infection
,
we
examined viral growth kinetics in
primary differentiated
human bronchial epithelial cells
(HBECs)
, grown
in
transwells on an air
-
liquid interface (ALI)
.
As expected, the N:G215C virus
produced the stable N dimer
in infected HBECs when harvested under non
-
reducing conditions
(
Fig
4
D
). Notably,
in multi
-
cycle growth curves
the
mNG N:G215C
virus
had
improved growth
kinetics
, with a
peak
titer
more than 100 times greater
than
the
mNG WT
virus
(
Fig
4
E
)
.
Th
ese
.
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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;
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9
data suggest that the stably dimerized form of N conveys a particular advantage
to viral
replication
in
primary differentiated human
bronchial cells.
Next, we determined the effect of the N:G215C mutation
in vivo
in the Syrian Golden
Hamster model of
SARS
-
CoV
-
2
infection.
Three to four
week
-
old male hamsters were
inoculated intranasally with either PBS (mock),
10
4
PFU of
the WT neon green reporter SARS
-
CoV
-
2 (mNG WT) or
10
4
PFU of
the
neon green reporter SARS
-
CoV
-
2 containing the N:G215C
mutation (N:G215C). Animals were monitored for weight gain/loss daily
for seven days
, and
cohort
s
of five animals underwent nasal washing followed by euthanasia
to
obtain tissues to
determine viral loads in the lung at both day 2 and day 4 (Fi
g. 5A
).
On day seven, surviving
animals were euthanized and
lung
tissue collected for
virological and
histopathological analysis
.
While animals infected with the N:G215C mutation showed
significant
weight loss
, relative to
control WT virus, the kinetics were delayed with peak disease achieved at day 5
-
6 post
infection, 1
-
2 days after WT infection
(Fig
5
B
).
Strikingly,
despite
delayed
weight loss, viral
replication
was increased with the G215C mutation
.
M
odest but significant increases in
viral
titers were observed in the
nasal washes at day 4
,
and a sustained 10
-
fold increase
over WT
titers
in the lungs throughout the infection (Fig
5
C
-
D
).
Together, despite the kinetic delay, the
N:G215C mutant caused similar overall weight loss and augmented viral replication.
Examining histopathology
,
the N:G215C mutant
had
modest changes in
antigen
staining, but increased infiltration and damage relative to
WT
control
virus.
At day 2, both WT
and N:G215C had similar antigen distribution and scores
(Fig.
S3
A
-
B
)
.
By
day 4,
N:G215 had
a modest
increase
in
overall antigen staining mostly driven by
significant
differences in airway
staining
. Viral staining was cleared in both WT and mutant infected animals by day 7 post
infection.
Examining immune infiltration and damage, lesions were of similar composition and
size at day 2 for both groups
, but more severe in WT animals
(Fig. 5E
-
F)
. However, at day 4,
N:G215C had increased
infiltration and damage
compared to WT infected animals. While
interstitial pneumonia, bronchiolitis, periarterial edema was
common in both groups, N:G215C
.
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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;
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doi:
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infected mice were consistently observed to have epithelial cytopathology and subendothelial
mononuclear cells. Similarly at day 7, N:G215C maintained evidence of significant damage
with
continued peribrochiolitis and epithelial cytopathology; in contrast, WT infected hamsters had
reduced overall damage scores. Together, the results demonstrate that despite similar weight
loss, the N:G215C mutant infected animals have increased viral
antigen accumulation and
damage in the lung as compared to control.
Wildtype SARS
-
CoV
-
2 containing the G215C mutation packages
more nucleocapsid
protein per virion
and
displays
oblong morphology
The SARS
-
CoV
-
2 nucleocapsid, like nucleocapsids of other Betacoronaviruses, is a
highly multifunctional protein.
Like other coronavirus nucleocapsids, it is thought to play key
roles
in the packaging of viral RNA
25
, the production of viral RNA through interactions with the
replication
-
transcription complex
10
13
,
and the antagonism of the innate immune response
26
28
.
To
better
understand why th
e N: G215C
mutation was important at a molecular level, we looked
at
where in the viral life
cycle the stably dimerized form of N was observed.
We first observed
the stable N
-
dimer in lysates
of
cells infected with the Beta, Delta and Iota variants
(Fig
2
A)
,
though cells transfected with the Delta (and to lesser extent Iota) nucleocapsids were able to
form the durable N
-
dimer even in the absence of other viral machinery (
Fig
3
A, B). As we had
observed a gradient of dimer formation depending on where the cysteine mutation was located
(215>243>185
; Fig
2
B,
3
B
),
we next explored whether the stable G215C N
-
dimer was found
at
highest levels
in transfected cells, infected cells, or in concentrated extracellular virions.
Accordingly, we measured the ratio of
N dimer:N monomer visible on a western blot of samples
collected under non
-
reducing conditions from transfected HEK
-
293T cells, infected Vero
-
TMPRSS2 cells, or
extracellular virus concentrated by polyethyl
ene glycol (PEG) precipitation
(Fig
6
A
-
C)
.
Interestingly, we noted that the dimer was enriched in
extracellular virus (Fig
6
C
, D
)
,
.
CC-BY-NC-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted March 11, 2024.
;
https://doi.org/10.1101/2024.03.08.584120
doi:
bioRxiv preprint