Proteases of the respiratory tract activating influenza viruses: Identification, characterization and cellular compartmentalization

Prof. Dr. Wolfgang Garten

Institute of Virology
Hans-Meerwein-Str. 2
35043 Marburg

E-mail: garten@staff.uni-marburg.de
Phone: +49 6421 286 5145
Homepage: http://www.uni-marburg.de/fb20/virologie/forschung/researchviro/gartenind

Dr. Eva Friebertshäuser

Institute of Virology
Hans-Meerwein-Str. 2
35043 Marburg

E-mail: friebertshaeuser@staff.uni-marburg.de
Phone: +49 6421 286 6019

Project description:

Human influenza A viruses cause acute respiratory illness that affects millions of people during seasonal outbreaks and occasional pandemics. Pathogenicity of influenza A viruses is determined by many factors, however, proteolytic cleavage of its major surface glycoprotein hemagglutinin (HA) plays an important role.

The HA mediates both binding to cell surface receptors and fusion of viral and endosomal membranes following endocytosis in order to release the viral genome into the cytoplasm of the target cell. HA is synthesized as a precursor protein HA0 that has to be cleaved by a host cell protease into the disulfide linked subunits HA1 and HA2 to gain its fusion capacity (Fig. 1). Proteolytic cleavage of HA is prerequisite to undergo conformational changes at low pH that expose the N-terminal hydrophobic fusion peptide of HA2 and trigger membrane fusion, and is therefore essential for viral infectivity and spread.

Highly pathogenic avian influenza A viruses of subtypes H5 and H7 possess a HA with multibasic cleavage site that is cleaved by ubiquitous endoproteases furin and PC6, supporting a systemic spread of viral infection (Fig. 1B). In contrast, HA of mammalian and low pathogenic avian influenza A viruses contains a single arginine, rarely a single lysine, at the cleavage site. In general, activation of HA0 with such a monobasic cleavage site was assumed to occur extracellularly when virions are already released from the cells, and trypsin and several “trypsin-like” proteases have been identified as HA-activating enzymes in vitro. Relevant HA-activating proteases in the human airway epithelium were only poorly defined until recently.

Fig. 1: A) Structure of the HA0 monomer and cleaved HA (Chen et al. 1998). Cleavage site: yellow, HA1: blue, HA2: red. B) Cleavage of HA0 into the disulfide linked subunits HA1 and HA2 at a specific cleavage site. TM: transmembrane domain.

We have established that the serine proteases HAT (human airway trypsin-like protease) and TMPRSS2 (transmembrane protease, serine S1 member 2) known to be present in the human airway epithelium cleave HA containing a monobasic cleavage site. Both are type II transmembrane proteases and recently we demonstrated that HA is cleaved by membrane-bound TMPRSS2 and HAT and differs in subcellular localization: activation of HA by TMPRSS2 occurs intracellularly, whereas HAT is enzymatically active at the surface of cells (Fig.2).

Fig. 2) Model of proteolytic activation and spread of influenza A virus in the human airway epithelium. Ciliated and non-ciliated cells of the epithelium are shown. The HA-activating proteases are shown by scissors: HAT (blue), TMPRSS2 (red); continuous arrows indicate infection of cells; dashed arrows release of virus progeny. (A) HAT- and TMPRSS2- expressing cells are productively infected with virus and spread infectious virus (green). (B) Infectious virus enters cells without suitable proteases: production of non-infectious viruses (black); HA is not cleaved. (C) Virus with non-cleaved HA is unable to infect cells only expressing TMPRSS2 or cells without expression of any relevant protease. (D) Infection of HAT-expressing cells with virus containing non-cleaved HA (black) is supported by HA cleavage on virions prior to virus entry. Infectious virus progeny are released. (Böttcher-Friebertshäuser et al., 2010)

The aims of our studies are now:

• Detailed subcellular localization and expression of TMPRSS2 and HAT
• Unravelling of molecular mechanisms underlying differences in proteolytic activities of HAT and TMPRSS2 on the cell surface, in cell supernatants and within cells
• Screening for further HA-activating proteases and airway proteases that are not capable of cleaving HA

Staff:

Dr. Eva Friebertshäuser, Postdoc
Catharina Freuer, PhD student
Carolin Tarnow, PhD student
Kerstin Pawletko, BSc student
Carolin Baum, BSc student
Petra Neubauer-Rädel, Technician

Selected publications since 2007:

1. Klenk HD, Garten W, Matrosovich M. (2011) Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic. Bioessays. 33:180-188.
2. Böttcher-Friebertshäuser E, Stein DA, Klenk HD, Garten W. (2011) Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin activating protease TMPRSS2. J Virol. 85:1554-1562.
3. Böttcher-Friebertshäuser E, Freuer C, Sielaff F, Schmidt S, Eickmann M, Uhlendorff J, Steinmetzer T, Klenk HD, Garten W. (2010) Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. J Virol. 84:5605-5614.
4. Okumura Y, Takahashi E, Yano M, Ohuchi M, Daidoji T, Nakaya T, Böttcher E, Garten W, Klenk HD, Kido H. (2010) Novel type II transmembrane serine proteases, MSPL and TMPRSS13, proteolytically activate membrane fusion activity of hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication. J Virol. 84:5089-5096.
5. Schlie K, Maisa A, Freiberg F, Groseth A, Strecker T, Garten W. (2010b) Viral protein determinants of Lassa virus entry and release from polarized epithelial cells. J Virol. 84:3178-3188.
6. Becker GL, Sielaff F, Than ME, Lindberg I, Routhier S, Day R, Lu Y, Garten W, Steinmetzer T. (2010) Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics. J Med Chem. 53:1067-1075.
7. Schlie K, Maisa A, Lennartz F, Ströher U, Garten W, Strecker T. (2010) Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication. J Virol. 84:983-992.
8. Böttcher E, Freuer C, Steinmetzer T, Klenk HD, Garten W. (2009) MDCK cells that express proteases TMPRSS2 and HAT provide a cell system to propagate influenza viruses in the absence of trypsin and to study cleavage of HA and its inhibition. Vaccine. 27:6324-6329.
9. Maisa A, Ströher U, Klenk HD, Garten W, Strecker T. (2009) Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1)-antitrypsin variants. PLoS Negl Trop Dis. 3(6):e446.
10. Garten W and HD Klenk. (2008) Cleavage Activation of the influenza virus hemagglutinin and its role in pathogenesis. In: Avian Influenza. Ed. HD Klenk, MN Matrosovich, J Stech. Monogr. Virol. Basel, Karger, vol 27, pp 156-167.
11. Böttcher E, Matrosovich M, Matrosovich T, Klenk HD, Garten W. (2007) Studies on proteolytic activation of human influenza viruses in stable cell lines expressing serine proteases TMPRSS2 and HAT. In Options for the Control of Influenza VI, pp.525-527. Edited by JM Katz. International Medical Press.