Ubiquitination Upregulates Influenza Virus Polymerase Function.
The influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation.IMPORTANCEThe cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.
Graduate Program in Cellular & Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Medical Microbiology & Immunology, University of Wisconsin-Madison Madison, Wisconsin, USA.
James
J
Kirui
Medical Microbiology & Immunology, University of Wisconsin-Madison Madison, Wisconsin, USA.
Arindam
A
Mondal
Medical Microbiology & Immunology, University of Wisconsin-Madison Madison, Wisconsin, USA amehle@wisc.edu.
Andrew
http://orcid.org/0000-0001-6060-4330
A
Mehle
Virus Res. 2011 Jun;158(1-2):12-27
21376763
Annu Rev Biochem. 1998;67:425-79
9759494
Cell Host Microbe. 2008 Aug 14;4(2):111-22
18692771
J Virol. 2013 Apr;87(8):4523-33
23408607
Nature. 2009 Apr 16;458(7240):914-8
19194459
EMBO J. 2010 Nov 17;29(22):3879-90
20924359
J Virol. 1997 Dec;71(12):9690-700
9371635
PLoS Pathog. 2013 Mar;9(3):e1003275
23555270
J Virol. 2014 Aug;88(16):9379-90
24920808
Nat Commun. 2013;4:1591
23481399
Nature. 2009 Apr 16;458(7240):909-13
19194458
PLoS Pathog. 2015 Aug 11;11(8):e1005095
26263374
J Virol. 2014 Jun;88(11):5977-86
24623411
J Virol. 1993 Apr;67(4):1761-4
8445709
Proc Natl Acad Sci U S A. 2015 Nov 10;112(45):14048-53
26504237
Acta Virol. 2013;57(2):113-22
23600869
Nat Struct Mol Biol. 2008 May;15(5):500-6
18454157
PLoS Pathog. 2012;8(11):e1002993
23144613
Curr Opin Virol. 2016 Apr;17:1-10
26426962
Proc Natl Acad Sci U S A. 2013 Oct 22;110(43):17516-21
24101521
J Gen Virol. 2009 Jun;90(Pt 6):1398-407
19264657
J Virol. 2010 Sep;84(18):9625-31
20631148
PLoS Pathog. 2015 Jun 09;11(6):e1004960
26057645
J Virol. 2013 Dec;87(24):13321-9
24089552
J Virol. 2015 Jun;89(11):5822-34
25787277
Virology. 2005 Oct 10;341(1):34-46
16084555
Traffic. 2003 Dec;4(12 ):857-68
14617349
J Virol. 2010 Oct;84(20):10477-87
20702645
Cell Host Microbe. 2010 Jun 25;7(6):427-39
20542247
PLoS Pathog. 2015 Apr 13;11(4):e1004826
25867750
Annu Rev Biochem. 2012;81:203-29
22524316
J Vis Exp. 2009 Aug 19;(30):null
19692941
J Virol. 2015 Jan 15;89(2):1452-5
25355893
Science. 2014 Oct 24;346(6208):473-7
25342804
J Biol Chem. 2004 Feb 27;279(9):7792-8
14672928
PLoS Pathog. 2008 Jun 06;4(6):e1000085
18535660