TY - JOUR
T1 - A sensitive yellow fever virus entry reporter identifies valosin-containing protein (VCP/P97) as an essential host factor for flavivirus uncoating
AU - Ramanathan, Harish N.
AU - Zhang, Shuo
AU - Douam, Florian
AU - Mar, Katrina B.
AU - Chang, Jinhong
AU - Yang, Priscilla L.
AU - Schoggins, John W.
AU - Ploss, Alexander
AU - Lindenbach, Brett D.
N1 - Funding Information:
This study was supported by PHS grants AI087925, AI131518, and AI120113 (all to B.D.L.) and AI107301 (to. A.P.) and by a Burroughs-Wellcome Fund Investigator in Pathogenesis Award (101539) to A.P.
Funding Information:
We thank Charles M. Rice (The Rockefeller University) for providing pACNR/YF17D, pACNR/YF?SK, pSINrep21/YF NS1, pCC1/YF17D, and pYF-17D/5'C25Venus2AUbi; Marie Flamand (Institut Pasteur) for providing NS1-specific monoclonal antibodies; Sergei Kotenko (Rutgers University) for providing C57BL/6J IFNAR?/? mice; Doug Brackney (Connecticut Agricultural Experiment Station) for providing BHK-21 clone 15 cells; and Christian Schlieker for providing pcDNA3.1(+)/VCP/p97-WT and pcDNA3.1(+)/VCP/ p97-QQ. This study was supported by PHS grants AI087925, AI131518, and AI120113 (all to B.D.L.) and AI107301 (to. A.P.) and by a Burroughs-Wellcome Fund Investigator in Pathogenesis Award (101539) to A.P.
Publisher Copyright:
© 2020 Ramanathan et al.
PY - 2020/3/1
Y1 - 2020/3/1
N2 - While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replicationdefective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin-and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
AB - While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replicationdefective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin-and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
KW - Flavivirus
KW - Nucleocapsid
KW - Uncoating
KW - Viral entry
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U2 - 10.1128/mBio.00467-20
DO - 10.1128/mBio.00467-20
M3 - Article
C2 - 32291299
AN - SCOPUS:85083408755
SN - 2161-2129
VL - 11
JO - mBio
JF - mBio
IS - 2
M1 - e00467-20
ER -