Science - USA (2021-12-24)

SCIENCEscience.org 24 DECEMBER 2021¥VOL 374 ISSUE 6575 1627

M

IRES

E

Luc

+T20

ORF1ab S EM N

gRNA

T20

SARS-CoV-2

293T 293T

ACE2/TMPRSS2

SC2-VLPs

HO

N

S

N

S

OH

O

VLPs

ΔS ΔM ΔN ΔE

ΔT20

0

5000

10000

15000

12757

17 22 17 59 204

12757

17 22 17 59 204

RLU

VLPs

ΔM

2xStrep-MM-2xStrep

Blank

0

10000

20000

30000

35 290 47 33

24318

35 290 47 33

RLU

Load onto 10-40%

sucrose gradient

Centrifuge - SW41

28000 RPM x 3hrs

Collect fractions

0.01 0.1 1 10 100

0

10000

20000

30000

40000

ng spike plasmid

RLU

48

100

180

αSpike

αN

S

NS

S2

SC2-VLPsΔS ΔM ΔN ΔE ΔT202xStrep-MM-2xStrep100X SpikeSC2-VLPs

Collect/filter

supernatant

Centrifuge - SW28

28000 RPM x 2hrs

Collect VLPs in pellet

Load onto 20%

sucrose cushion

Filtered

Supernatant

1 2 3 4 5 6 7 8 9 10 11 blank

0

5000

10000

15000

20000

RLU

Sedimentation

Spike

0.33 0.67

0.0016 1.0

N

M

IRES

E

Luc

+T20

Spike

N

Light

S

M

E

N

gRNA

Reporter+T20

SC2-VLPs

A

B

CD

F G

E

H

I

Fig. 1. Design and characterization of SC2-VLPs.(A) Schematic of SARS-CoV-2
and SC2-VLP design and location of RNA packaging sequence T20. (B) Process
for generating and detecting luciferase-encoding SC2-VLPs. Numbers below plasmid
maps indicate ratios used for transfection. (C) Induced luciferase expression
measured in receiver cells (293T-ACE2/TMPRSS2) from“standard”SC2-VLPs
containing S, M, N, E, and luciferase-T20 transcript, as well as VLPs lacking each one
of the components. (D) N- or C-terminal 2× Strep-tag on M abrogates vector-induced
luciferase expression. (E) Optimal luciferase expression requires a narrow range

of spike plasmid concentrations corresponding to^1 = 500 of the total plasmid mass used

for transfection. (F) Schematic for purification of SC2-VLPs. (G) Western blot

showing S and N in pellets purified from standard SC2-VLPs and conditions that did

not induce luciferase expression in receiver cells. (H) Schematic for sucrose gradient

for separating SC2-VLPs. (I) Induced luciferase expression from sucrose gradient

fractions of SC2-VLPs. gRNA, genomic RNA; IRES, internal ribosome entry site;

Luc, luciferase; RLU, relative light units; S2, cleavage product of S; NS, nonspecific

band. Error bars indicate SD withN= 3 independent transfections in each case.

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