Science - USA (2021-12-24)

in our work provides structural evidence for
the RNA channeling hypothesis ( 20 ). How-
ever, contradictory to previous assumptions
that Pol IV RNA has to be completely re-
leased from the Pol IV RNA exit channel in
a5′-to-3′direction to expose the 3′terminus
for RDR2 engagement ( 20 , 51 ), we found that
the Pol IV RNA is threaded inside of the two-
RNA-polymerase complex through the inter-
polymerase RNA channel in a 3′-to-5′direction
to the RDR2 active center, where the 3′terminus
of Pol IV RNA is directly captured and used
as the template for RDR2 RNA synthesis.
The backtracking-triggered RNA channeling
model suggests that RDR2 pulls and releases
Pol IV RNA in a 3′to 5′direction from the Pol
IV secondary channel, and thus represents a
previously undescribed means of transcription
termination by Pol IV, a dsRNA-induced ter-
mination (Fig. 6). This termination mode is
distinct from all previous transcription termi-
nation models ( 52 , 53 ). The dsRNA-induced
termination model predicts that Pol IV RNA
production should be hindered if Pol IV is un-
coupled from RDR2; this prediction is sup-
ported by previous in vivo RNA-seq results
which showed that Pol IV RNA production is
impaired in RDR2 mutants ( 6 , 8 , 20 ), and by
a previous in vitro transcription study which
showed that Pol IV is unable to terminate
properly if RDR2 RNA transcripts are not
detected ( 20 ).

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ACKNOWLEDGMENTS

We thank T. Xu for theA. thalianaT87 cell line, L. Kong, F. Wang,

G. Li, and J. Duan at the cryo-EM center of the National Center

for Protein Science Shanghai; B. Zhu and X. Li at the center for

Biological Imaging of the CAS Institute of Biophysics; S. Chang at

the cryo-EM center of Zhejiang University; and X. Ma at the

cryo-EM center of SUST for assistance with data collection.Funding:

J.W. received National Natural Science Foundation of China Grant

31788103 and Strategic Priority Research Program of CAS grant

XDB27030101; Y.Z. received the Shanghai Pilot Program for

Basic Research–Chinese Academy of Science, Shanghai Branch,

National Natural Science Foundation of China grant 31822001,

and Leading Science Key Research Program of CAS grant QYZDB-

SSW-SMC005.Author contributions:Conceptualization: Y.Z.

and J.W. Pol IV preparation: K.H., C.Z., J.G., H.Z., Z.G., and W.M.

Structure determination: K.H., Y.F., C.F., X.W., and L.Y. Transcription

assay: C.F. and K.H. RNA and DNA sequencing: Z.X. and K.H.

Funding acquisition: Y.Z., J.W., and Y.F. Project administration:

Y.Z., J.W., and Y.F. Supervision: Y.Z., J.W., and Y.F. Writing–original

draft: Y.Z. and J.W. Writing–review and editing: Y.Z., J.W., Y.F.,

K.H., X.W. and C.F.Competing interests:Authors declare that they

have no competing interests.Data and materials availability:

The cryo-EM map and coordinates were deposited in Protein

Data Bank and Electron Microscopy Data Bank (Pol IV–RDR2

holoenzyme: 7EU1 and EMD-31306; Pol IV–RDR2 bTEC: 7EU0 and

EMD-31305). The small RNA-seq and bisulfite-seq data (BioProject

PRJCA005428) were deposited in the Beijing Institute of Genomics

Data Center (https://ngdc.cncb.ac.cn/).

SUPPLEMENTARY MATERIALS

science.org/doi/10.1126/science.abj9184

Materials and Methods

Figs. S1 to S12

Tables S1 to S3

References ( 54 – 66 )

MDAR Reproducibility Checklist

Movies S1 to S5

14 June 2021; accepted 9 November 2021

10.1126/science.abj9184

CORONAVIRUS

An oral SARS-CoV-2 M

pro

inhibitor clinical candidate

for the treatment of COVID-19

Dafydd R. Owen^1 *, Charlotte M. N. Allerton^1 , Annaliesa S. Anderson^2 , Lisa Aschenbrenner^3 ,

Melissa Avery^3 , Simon Berritt^3 , Britton Boras^4 , Rhonda D. Cardin^2 , Anthony Carlo^3 ,

Karen J. Coffman^3 , Alyssa Dantonio^3 , Li Di^3 , Heather Eng^3 , RoseAnn Ferre^4 , Ketan S. Gajiwala^4 ,

Scott A. Gibson^5 , Samantha E. Greasley^4 , Brett L. Hurst^5 , Eugene P. Kadar^3 , Amit S. Kalgutkar^1 ,

Jack C. Lee^3 , Jisun Lee^3 , Wei Liu^4 , Stephen W. Mason^2 †, Stephen Noell^3 , Jonathan J. Novak^3 ‡,

R. Scott Obach^3 , Kevin Ogilvie^3 , Nandini C. Patel^1 , Martin Pettersson^1 ¤, Devendra K. Rai^2 ,

Matthew R. Reese^3 , Matthew F. Sammons^1 , Jean G. Sathish^2 , Ravi Shankar P. Singh^1 ,

Claire M. Steppan^3 , Al E. Stewart^4 , Jamison B. Tuttle^1 , Lawrence Updyke^1 , Patrick R. Verhoest^1 ,

Liuqing Wei^3 , Qingyi Yang^1 , Yuao Zhu^2

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an

important part of the healthcare response to countering the ongoing threat presented by COVID-19.

Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2

main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target

selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted

SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell

potency in a phase 1 clinical trial in healthy human participants.

H

uman coronavirus infections are com-

mon, with at least four examples (229E,

NL63, OC43, and HKU1) now consi-

dered endemic ( 1 ). However, the emer-

gence within the past 20 years of severe

acute respiratory syndrome coronavirus

1 (SARS-CoV-1), Middle East respiratory

syndrome coronavirus (MERS-CoV), and

SARS-CoV-2 as novel human coronaviruses

has signaled the substantial threat poten-

tial of this viral class. The catastrophic

SARS-CoV-2 outbreak of 2019 has resulted

1586 24 DECEMBER 2021•VOL 374 ISSUE 6575 science.orgSCIENCE

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