Interactions of the influenza virus polymerase and hemagglutinin with host factors and their roles in pathogenesis and host adaptation
Prof. Dr. Hans Dieter Klenk
Institut für Virologie
Philipps-Universität Marburg
Hans-Meerwein Str. 2
35043 Marburg
Fon: +49-6421-286 6191
+49 6421-286 6254 (Sek)
Fax: +49-6421-286 8962
E-Mail: Klenk@staff.uni-marburg.de
Homepage:
www.uni-marburg.de/fb20/virologie/forschung/researchviro/klenk
Project description
Host range and pathogenicity of Influenza A virus are polygenic
traits depending on the interaction of different viral proteins with
specific host factors. Evidence is increasing that such interactions
play an important role when the virus crosses borders between different
compartments of the host cell. The viral polymerase, known to be a
major determinant of host range, has to enter into the nucleus of the
infected cell in order to promote replication and transcription of the
viral genome. In a comparative study of an avian influenza strain and
its mouse adapted variant we have shown that adaptation to mice
depended exclusively on mutations in the polymerase proteins. These
findings supported the concept that adaptation of the polymerase to
host factors is an important mechanism underlying interspecies
transmission. In the last funding period we have identified importin
α1, a component of the nuclear pore complex, as such a host factor.
Adaptive mutations in polymerase subunits improve binding to importin
α1 in mammalian cells. As a result, nuclear transport of these proteins
and efficiency of replication are enhanced. We could also show that the
adapted virus spreads systemically in mice, whereas the non-adapted
virus causes local infection in the respiratory tract. Thus, the
interaction of the viral polymerase with the nuclear import machinery
is an important determinant of host range and pathogenicity. Some of
the structural features typical for avian viruses have been preserved
in the polymerase of the 2009 pandemic influenza A virus (H1N1v) and in
H5N1 viruses occasionally transmitted to man, suggesting that these
viruses have the potential to further adapt to humans and that their
pathogenic and pandemic potential is not exhausted yet. In the next
funding period, we would like to prove this concept by generating
mutants and reassortants of H1N1v virus containing modified polymerase
genes and by analyzing their replicative and pathogenic properties.
Hemagglutinin activation by host proteases residing in the TGN and the
plasma membrane is also an important determinant of pathogenicity.
Avian H9N2 viruses that have occasionally been transmitted to humans
and may therefore cause a future pandemic have a polybasic
hemagglutinin cleavage site typical for highly pathogenic viruses, but
they do not show the high cleavability required for high pathogenicity.
We assume that minor structural changes of the cleavage site might lead
to high pathogenicity. To prove this concept we will generate H9N2
protease activation mutants and analyze their replicative and
pathogenic properties.
Joanna Baron (PhD student)
Folker Schwalm (PhD student)
Publications since 2007
1. Matrosovich, M., Matrosovich, T., Uhlendorff, J., Garten, W., and Klenk, H.-D.: Avian-virus like receptor specificity of the hemagglutinin impedes influenza virus replication in cultures of human airway epithelium. Virology 361, 384-390 (2007).2. Runkler, N., Lehmann, C., Schneider-Schaulies, S., Klenk, H.-D., and Maisner, A.: Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein. Cellular Microbiology 9, 1203-1214 (2007).
3. Zhirnov, O. P., Vorobjeva, I. V., Saphonova, O. A., Malyshev, N. A., Ovcharenko, A. V.and Klenk, H.-D.: Biochemical properties of clinical influenza viruses propagated in human intestinal cell line. Biochimie 72, 493-505 (2007) (Russian).
4. Zhirnov, O. P., Vorobjeva, I. V., Saphonova, O. A., Malyshev, N. A., Ovcharenko, A. V.and Klenk, H.-D.: Specific biochemical features of replication of clinical influenza viruses in human intestinal cell culture. Biochemistry (Mosc) 72, 398-408 (2007).
5. Zhirnov, O. P. and Klenk, H.-D.: Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling. Apoptosis 12, 1419-1432 (2007).
6. Zhirnov O.P., Poyarkov, S.V., Vorobjeva, I.V., Safonova, O.A., Malyshev, N.A., and Klenk, H.-D.: Segment NS of influenza A virus contains an additional gene NSP in positive-sense orientation. Dokl Biochem Biophys 414, 127-133 (2007).
7. Klewitz, C., Klenk, H.-D. and ter Meulen, J.: Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity. J. Gen. Virol. 88, 2320-2328 (2007).
8. Jones, S., Ströher, U., Fernando, L., Qiu, X, Alimonti, J., Melito, P., Bray, M., Klenk, H.-D., and Feldmann, H.: Assessment of a vesicular stomatitis virus based vaccine using the mouse model of Ebola virus hemorrhagic fever. J. Inf. Dis. 196 (Suppl. 2), 404-412 (2007).
9. Gabriel, G., Abram, M., Keiner, B., Wagner, R., Klenk, H.-D., and Stech, J.: Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J. Virol. 81, 9601-04 (2007) .
10. Runkler, N., Pohl, C., Schneider-Schaulies, S., Klenk H.-D, and Maisner, A.: Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein. Cell Microbiol. 9, 1203-1214 (2007).
11. Gabriel, G., Garn, H., Weymann, M., Renz, H., Herwig, A., Klenk, H.-D., and Stech, J.: The potential of a protease activation mutant of a highly pathogenic avian influenza virus for a pandemic live vaccine. Vaccine, 26, 956-965 (2008).
12. Zhirnov, O.P., Syrtsev, V.V., Vorobjeva, I.V., and Klenk, H.-D.: Site-directed modification of caspase cleavage site regions in avian influenza virus proteins. Vopr. Virusol. 53,16-21 (2008).
13. Gabriel, G., Herwig, A., and Klenk, H.-D.: Interaction of polymerase subunits PB2 and NP with importin α1 is a determinant of host range of influenza A virus. PloS Pathogens 4 (2): e11.doi: 10.1371/journal.ppat.0040011 (2008).
14. Gabriel, G., Nordmann, A., Stein, D.A., Iversen, P.L., and Klenk, H.-D.: Morpholino oligomers targeting PB1 and NP genes enhance survival of mice infected with highly pathogenic influenza A H7N7 virus. J. Gen. Virol. 89, 939-948 (2008)
15. Runkler, N., Dietzel, E., Moll, M., Klenk, H.-D., and Maisner, A.: Glycoprotein targeting signals influence distribution of measles virus envelope proteins and virus spread in lymphocyes. J. Gen. Virol. 89, 687-696 (2008).
16. Stech, J., Stech, O., Herwig, A., Altmeppen, H., Hundt, I., Gohrbrandt, S., Kreibich, A., Klenk, H.D., and Mettenleiter, T.C.: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acid Research, 2008, 1-6, doi:10.1093/nar/gkn646.
17. Matrosovich, M., Stech, J., and Klenk, H.D.: Influenza receptors, polymerase and host range, OIE Scientific and Technical Review 28 (1), 203-217 (2009).
18. Szecsi, J., Gabriel, G., Edfeldt, G., Michelet, M., Klenk, H.D., and Cosset, F.L.: DNA vaccination with a single-plasmid construct coding for virus-like particles protects mice against infection with a highly pathogenic avian influenza A virus. J. Inf. Dis. 200, 181-190 (2009).
19. Gabriel, G., Klingel, K., Planz, O., Bier, K., Herwig, A., Sauter, M., and Klenk, H.D.: Spread of infection and lymphocyte depletion in mice depends on polymerase of influenza virus. Am. J. Pathol. 175, 1178-1186 (2009).
20. Uhlendorff, J., Matrosovich, T., Klenk, H.D., and Matrosovich, M.: Functional significance of the hemadsorbing activity of influenza virus neuraminidase and its alteration in pandemic viruses. Arch. Virol. 154, 945-957 (2009).
21. Böttcher, E., Freuer, C., Steinmetzer, T., Klenk, H.D., and Garten, W.: 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 (2009).
22. Maisa, A., Ströher, U., Klenk, H.D., Garten, W., and Strecker, T.: Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted α1-antitrypsin variants. PLoS neglected Tropical Diseases, 3, e446 (2009).
23. Zhirnov, O.P., Klenk, H.D.: Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus. Virology 394, 57-63 (2009).
24. Okumura, Y., Takahashi, E., Yano, M., Ohuchi, M., Daidoji, T., Nakaya, T., Böttcher, E., Garten, W., Klenk, H.D., and Kido, H.: Novel type II transmembrane serine proteases, MSPL and TMPRSS13, Proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication. J. Virol. 84, 5089-5096 (2010).
25. Böttcher-Friebertshäuser, E., Freuer, C., Sielaff, F., Schmidt, S., Eickmann, M., Uhlendorff, J., Steinmetzer, T., Klenk, H.D., and Garten, W.: 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 (2010).
26. Liu, Y., Childs, R.A., Matrosovich, T., Wharton, S., Palma, A.S., Chai, W., Daniels, R., Gregory, V., Uhlendorff, J., Kiso, M., Klenk, H.D., Hay, A., Feizi, T. and Matrosovich, M.: Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J. Virol. 84, 12069-12074 (2010).
27. Keiner, B., Maenz, B., Wagner, R., Cattoli, G., Capua, I. and Klenk, H.D.: Intracellular distribution of NS1 correlates with the infectivity and interferon antagonism of an avian influenza virus (H7N1). J. Virol. 84, 11858-65 (2010).
28. Zhirnov, O.P. and Klenk, H.D.: [Integration of influenza A virus NSP gene into baculovirus genome and its expression in insect cells]. Vopr Virusol 55(2), 4-8 (2010).
29. He, Y., Xu, K., Keiner, B., Zhou, J., Czudai, V., Li, T., Chen, Z., Liu, J., Klenk, H.D., Shu, Y.L. and Sun, B.: Influenza A virus replication induces cell cycle arrest in G0/G1 phase. J. Virol. 84, 12832-40 (2010).
30. Xu, K., Klenk, C., Liu, B., Keiner, B., Cheng, J., Zheng, B.J., Li, L., Han, Q., Wang, C., Li, T., Chen, Z., Shu, Y., Liu, J., Klenk, H.D. and Sun, B.: Modification of nonstructural protein 1 of influenza A virus by SUMO1. J. Virol. 85, 1086-98 (2011).
31. Boettcher-Friebertshauser, E., Stein, D.A., Klenk, H.D. and Garten, W.: 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, 1554-62 (2011).
32. Klenk, H.-D.: Influenzaviren auf dem Weg vom Tier zum Menschen. Pharm. unserer Zeit 40, 104-108. (2011)
33. Gabriel, G., Klingel, K., Otte, A., Thiele, S., Hudjetz, B., Arman-Kalcek, G., Sauter, M., Shmidt, T., Rother, F., Baumgarte, S., Keiner, B., Hartmann, E., Bader, M., Brownlee, G.G., Fodor, E. and Klenk, H.D.: Differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus. Nat. Commun. 2, 156 (2011).
34. Klenk, H.D., Garten, W. and Matrosovich, M.: Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic. Bioessays 33, 180-188 (2011).
35. Zhirnov, O.P., Matrosovich, T.Y., Matrosovich, M.N. and Klenk, H.D.: Aprotinin, a protease inhibitor, suppresses proteolytic activation of pandemic H1N1v influenza virus. Antiviral Chemistry and Therapy, in press.
Publications in monographs
1. Slenczka, W., and Klenk, H.-D.: Forty years of Marburg virus. J. Inf. Dis. 196, (Suppl. 2), 131-135 (2007)2. Klenk, H.-D.: Viren zwischen Tier und Mensch – Wirtswechsel und Pathogenität zoonotischer Erreger. Robert Koch Mitt. 31, 38-45 (2007).
3. Klenk, H.D., Matrosovich, M., and Stech, J.: Avian Influenza: molecular mechanisms of pathogenesis and host range. In: Animal Viruses: Molecular Biology (T.C. Mettenleiter, F. Sobrino, eds.), pp. 253-301, Caister Academic Press (2008).
4. Matrosovich, M.N.; Gambaryan, A.S., and Klenk, H.D.: Receptor specificity of influenza viruses and its alteration during interspecies transmission. In: Avian Influenza, Monographs in Virology 27 (H.D. Klenk, M.N. Matrosovich, J. Stech, eds), pp 134-155, Karger, Basel (2008).
5. Garten, W. and Klenk, H.D.: Cleavage activation of the influenza virus hemagglutinin and its role in pathogenesis. In: Avian Influenza, Monographs in Virology 27 (H.D. Klenk, M.N. Matrosovich, J. Stech, eds) pp 156-167, Karger, Basel (2008)
6. Heckler, R., and Klenk, H.-D.: Orthomyxoviren, Influenzaviren. In: Burkhardt, Mikrobiologische Diagnostik (B. Neumeister, H.K. Geiss, R.W. Braun, P. Kimmig, Eds) Georg Thieme Verlag, Stuttgart, New York, pp. 939-948 (2009).
7. Stech, J. and Klenk, H. D.: Application of cleavage activation mutants of influenza virus as live vaccines (P.R. Dormitzer et al., eds.) Birkhäuser Advances in Infectious Diseases, pp 321-330, Springer, Basel (2011).
