Science - USA (2021-12-24)

1628 24 DECEMBER 2021¥VOL 374 ISSUE 6575 science.orgSCIENCE

ORF1ab S EM N

gRNA

ORF1ab S

1

87

EM N

gRNA

vs no PSNormalized

1

1

2

28 SC2-VLPs

tiled segments

293T

ACE2/TMPRSS2

5000 10000 15000 20000 25000 29903

HO

N

S

N

S

OH

O

0

2

Normalized

vs T20

20000 23000

T20

PS9

PS2

PS11

PS580

T20 nts 20080-22222

PS9 nts 20080-21171

ORF1ab S

• PS T

(^1) T (^2) T (^3) T (^4) T (^5) T (^6) T (^7) T (^8) T 9
T^1
0
T^1
2
T^1
3
T^1
4
T^1
5
T^1
6
T^1
7
T^1
8
T^1
9
T^2
0
T^2
1
T^2
2
T^2
3
T^2
4
T^2
5
T^2
6
T^2
7
T^2
8
bl
an
k
0
2500
5000
7500
10000
12500
RLU
-P
S
PS580
T^2
0
PS
2
PS
9
PS
11
0
50000
100000
150000
RLU
M
IRES
E
Spike
N
Light
nsp14 nsp15nsp16
A
B
D EF
C
GF
P-P
S9 GFP
bl
an
k
0
1
2
3
4
5
% GF
P+
Fig. 2. RNA packaging into SC2-VLPs by SARS-CoV-2 sequences.(A) Arrayed
screen for determining the location of the optimal sequence for RNA packaging in
SC2-VLPs. Two-kilobase tiled segments of the genome were cloned into the
3 ′UTR of the luciferase plasmid. (B) Induced luciferase expression in receiver
cells by SC2-VLPs containing different tiled segments from the SARS-CoV-2
genome. (C) Heatmap visualization of the data from (B), showing the locations
of tiled segments relative to the SARS-CoV-2 genome. Color intensity indicates
luminescence of receiver cells for each tile normalized to expression for a luciferase
plasmid containing no insert. (D) Smaller segments of the genome were used to
locate the optimal RNA packaging sequence. (E) Heatmap visualization of the data
from (D). (F) Flow cytometry analysis of GFP expression in 293T ACE2/TMPRSS2
cells incubated with SC2-VLPs encoding GFP-PS9, GFP (no packaging sequence),
or no VLPs. nts, nucleotides. Error bars indicate SD withN= 3 independent
transfections in each case.
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