and a“delayed polymerase stalling”inhibitor
well characterized for SARS-CoV-2—along with
RSV and other RNA viruses ( 21 , 22 ), corrobo-
rated this antiviral effect of 4′-FlU-TP (Fig. 2E
and data S1).
When a modified template coded for incor-
poration of only a single UTP (Fig. 2F and data
S1), primers elongated preferentially to posi-
tioni +3 after4′-FlU-TP, whereas the effi-
ciency of full elongation was strongly reduced
compared with extension in the presence of
UTP. However, repositioning the incorpora-
tion site further downstream in the template
triggered immediate polymerase stalling at
positioni(fig. S5), indicating template se-
quence dependence of the inhibitory effect.
Transcription stalling atiori+ 3 was also
observed after multiple 4′-FlU incorporations:
An AxAxxx template (Fig. 2G) and direct tan-
dem incorporations through an AAxxAx tem-
plate (fig. S5) caused stalling at positioni,
whereas increasing spacer length between the
incorporated uridines shifted preferential stalling
toi+3 (fig. S5). This variable delayed polymerase-
stalling event within one to four nucleotides of
the incorporation site was equally prominent
when we examined de novo initiation of RNA
synthesis at the promoter with a synthetic native
RSV promoter sequence rather than extension of
primer-template pairs (fig. S6).
Purification of a core SARS-CoV-2 polymer-
ase complex [nonstructural proteins (nsp) 7, 8,
and12]frombacterialcelllysates( 23 , 24 ) (Fig.
2H) and assessment of RdRP bioactivity in
equivalent primer-extension in vitro polymer-
ase assays (Fig. 2I) again demonstrated incor-
poration of 4′-FlU-TP in place of UTP by the
coronavirus RdRP (Fig. 2J), but there was no
sign of immediate polymerase stalling. How-
ever, SARS-CoV-2 polymerase stalling was trig-
gered by multiple incorporations of 4′-FlU-TP,
and was particularly prominent when a sec-ond incorporation of 4′-FlU-TP occurred at the
i+4 position (Fig. 2K and fig. S7). Primer ex-
tension was blocked when the nsp12 subunit
was omitted or an nsp12 variant carrying muta-
tions in the catalytic site was used, confirming
specificity of the reaction (fig. S7 and data S1).4 ′-FlU is rapidly anabolized, metabolically
stable, and potently antiviral in disease-relevant
well-differentiated HAE cultures
Quantitation of 4′-FlU and its anabolites in
primary HAE cells (Fig. 3A) demonstrated
rapid intracellular accumulation of 4′-FlU,
reaching a level of 3.42 nmol/million cells in
the first hour of exposure (Fig. 3B). Anabolism
to bioactive 4′-FlU-TP was efficient, resulting
in concentrations of 10.38 nmol per million cells
at peak (4 hours after start of exposure) and
1.31 nmol per million cells at plateau (24 hours).
The anabolite was metabolically stable, remain-
ing present in sustained concentrations of164 14 JANUARY 2022•VOL 375 ISSUE 6577 science.orgSCIENCE
Fig. 4. Therapeutic oral efficacy of 4′-FlU
in the RSV mouse model.(A) Balb/cJ
mice were inoculated with recRSV-A2line19F-
[mKate] and treated as indicated. At
4.5 days after infection, viral lung titers
were determined with TCID 50 titration
(n= 5). (B) Balb/cJ mice were inoculated
with recRSV-A2line19F-[mKate] or mock-
infected, and treated as indicated. Blood
samples were collected before infection
and at 1.5, 2.5, 3.5, and 4.5 days after
infection; lymphocyte proportions with
platelets/ml are represented over time (n= 4).
(C) Balb/cJ mice were inoculated with
recRSV-A2line19F-[redFirefly] and treated
as indicated. In vivo luciferase activity
was measured daily. (D) Total photon flux
from mice lungs from (C) over time (n= 3).
(E) Balb/cJ mice were inoculated with
recRSV-A2line19F-[mKate] and treated as
indicated. At 4.5 days after infection, viral
lung titers were determined with TCID 50
titration (n= 5). In all panels, symbols
represent individual values, and bars or
lines represent means. One-way ordinary
analysis of variance (ANOVA) with Tukey’s
post hoc multiple comparisons (B) and (I) or
two-way ANOVA with Dunnett’sposthoc
multiple comparison (C) and (G). h.p.i.,
hours post-infection.p=0.0112
p=0.0025D vehicle
4’-FlU 5mg/kg +1 h.p.i.
4’-FlU 5mg/kg -24 h.p.i.*
**5
4
3
2
1Radiance (p/sec/cm2 /sr)min= 1.00e6 max= 1.00e7×10^6
+1 h.p.i.vehiclemockRSV mockRSV mockRSV-24 h.p.i.10
9
8
7
6RSV-A2line19F-[mKate]TCID/g lung tissue 50****B****
****p<0.0001vehicle
0.2 mg/kg1 mg/kg5 mg/kgRSV-A2line19F-[mKate]TCID/g lung tissue 50p<0.0001p<0.0001p<0.0001p=0.0006
p=0.0231vehicle2 h.p.i.12 h.p.i.24 h.p.i.36 h.p.i.48 h.p.i.****************Cdays post-infectionvehicle - mock
vehicle - RSV4’-FlU - mock
4’-FlU - RSVdays post-infectionplatelets/mln=55 mg/kg 4’-FlU / vehicle end of study RSV-A2line19F-[mKate] 5e05 TCID 50 (I.N.)2 h.p.i.n=5
12 h.p.i.n=5
24 h.p.i.n=5
36 h.p.i.n=5
48 h.p.i.n=5
vehicleEtime to failure studyday 0day 1day 2day 3day 4lung viral titern=3day 0
day 1
day 2
day 3
day 45 mg/kg 4’-FlU / vehicle
RSV-A2line19F-[redFirefly]
2.1e05 TCID 50 (I.N.)n=3n=3end of studyday 5bioluminescencein vivo visualization of RSV replicationday -1n=3Aday 0 n=5day 1day 2day 3day 40.2/1/5 mg/kg 4’-FlU / vehicle
RSV-A2line19F-[mKate]
5e05 TCID 50 (I.N.)lung viral titer0.2 mg/kg end of studyn=5
1 mg/kgn=5
5 mg/kgn=5
vehicledose to failure study101102103104105l.o.d.n=4day 0day 1day 2day 3day 4n=45 mg/kg 4’-FlU / vehicleRSV-A2line19F-[mKate] 3e05 TCID 50 (I.N.)blood draw end of studytolerability - hematologyn=4 n=4Lymphocytes (%) 012345
60708090100
p>0.05012345107108109p>0.05101102103104105l.o.d.0123456011082108days post-infectionphotons/sRESEARCH | RESEARCH ARTICLES