Concluding remarks and outlook - Cellular factors modulating the entry efficiency of West Nile

5 Discussion

5.5 Concluding remarks and outlook

The Discussion chapter clearly demonstrates that the present knowledge about WNV entry, the molecules, the mechanisms and the entry-induced signalling pathways involved, is far from being satisfactory. Findings provided by this study reiterate the complexity of the entry process found in other viruses. The major conclusions are as follows:

(i) The presence of αv, β1 or β3 integrins is not a requirement for a successful WNV infection of the cell lines used here (5.3.1.2). This conclusion is based on the fact that all four WNV strains were capable to replicate in the integrin deficient mouse fibro-blasts.

(ii) The early assumption that integrins function as receptors that mediate virus binding and internalisation in an integrated step is rejected (5.3.1.3). Virus attachment to cell surfac-es of integrin deficient cells is comparable to those of integrin exprsurfac-essing cells. Similar-ly, blocking antibodies against β1 and β3 integrin subunits failed to interfere with virus binding to integrin expressing cells and had no effect on the infection outcome.

(iii) Integrin expression positively affects virus yields (5.3.1.2), presumably at the level of virus internalisation, by enhanced endocytosis or by interacting with other unidentified surface receptors, either by physical contact or by integrin-mediated signalling (see also 5.3.1.6). It became clear in this study, however, that other entry routes are used by WNV instead or in parallel to the integrins in question.

(iv) Differences among WNV strains do not affect the usage of β1 and β3 integrins during entry and/or replication (5.3.1.5). While the strain plays a major role in virulence, infec-tivity and in the development of the disease results from this study indicate that effi-ciency of virus entry with respect to the involvement of integrins are, on the whole, the same for all strains used here.

(v) It is assumed that another surface protein different from integrin αvβ3 is necessary to induce virus internalisation as an integrin-independent process or in cooperation with integrin αvβ3 by joint signalling (5.4). Rescue of the β3 subunit in CHO-K1 cells and constitutive expression of integrin αvβ3 did not result in permissiveness of these cells.

Efficiency of binding to CHO-K1 was essentially the same as in other cell types.

(vi) Heparan sulfate obviously serves as a possible attachment factor for WNV but accounts for a limited share in virus attachment only (5.3.3). Binding to GAG- or specifically HS-deficient cells was slightly but significantly reduced.

These aspects have important implications for the current model of WNV pathogenesis in respect to entry mechanisms and susceptibility of cells. However, further studies are neces-sary designed to isolate the crucial receptors and to answer questions as to whether expression and functions of these receptors may influence WNV infection in vivo. Based on the results of this work the importance of integrin-mediated signalling for WNV entry has to be addressed in future studies:

• Additional efforts to achieve a successful rescue of a β3 subunit without its cytoplasmic domain are pivotal in order to make a decision on the necessity of the signalling cascade for virus uptake.

• Besides, the detection of pathways activated upon virus binding, i.e. activation of key proteins such as RhoA, Cdc42, Rac1, Ras, PI3-K and Rab (see Figure 47), that are con-nected to integrins and other signalling receptors, is important. It not only allows depicting the inside-directed signalling cascade related to virus infection but has also a great poten-tial in identifying putative receptor candidates connected to the activated signalling path-ways. Additionally, manipulation of the relevant host cell components by genetic and bio-chemical means may be helpful in this context.

• The application of the cell culture model used in this study should be extended to infection experiments with αv rescue MEFs, derived from the corresponding αv-deficient cells, to analyse the effect of αv integrin on WNV replication.

• Another interesting approach for further infection experiments may be to knock down both β1 and β3 subunits to investigate the additive or synergistic quality of the negative effect on WNV replication.

• The physical interaction between WNV and integrins should be clarified since the results yielded in this study differ from those achieved by Chu and Ng (5.3.1.3).

• Finally, CHO cells have a great potential since, as far as known, they constitute the only cell line resistant to WNV infection. Achieving permissiveness for WNV by substitution of an absent receptor protein by transfection of these cells would unequivocally identify it as such.

The knowledge of the general mechanisms of infection and replication of WNV is crucial for the control of this virus worldwide. An important question that was addressed in this study concerns the viral receptors as potential determinants not only for host and tissue tropism but also for pathogenesis. Soluble receptors and anti-receptor compounds constitute potential tools for control measures as they can prevent the interaction between the virus and its cellular receptor. The overall aim of this study was to spotlight the putative involvement of integrins as receptors in WNV entry.

In summary, the results obtained in this study corroborate the concept that WNV entry is a multifarious process in which the cooperation of several cellular surface proteins is required and where different molecules are used consecutively or alternatively for binding and endocy-tosis. The simple one-receptor-mediates-entry theory is unlikely to count for WNV infection of susceptible host cells. Integrins in particular are definitively involved in WNV infection of cells. However, it is unclear at which stage of virus entry and in which way.

Acknowledgements

I would like to express my warmest thanks to all colleagues and friends who substantially supported the realisation of this work.

I am most indebted to Prof. Dr. Martin H. Groschup for his outstanding support, the scientific encouragement I got from him, and especially his belief in me and my work. There is no telling how much my work profited from such an open intellectual atmosphere and stimulat-ing context. I greatly appreciate havstimulat-ing him as a supervisor as he was always open for discussion and critical arguments and showed an amazing competence in handling exigencies.

I would like to express my special appreciation to Dr. Markus Keller for supervising my thesis and for the introduction into the topic. He laid out the theoretical and methological basis of my work, and not only provided his great experiences in establishing MEFs but also his advice and expertise in many different fields. Numerous constructive discussions with him considerably contributed to the success of this work.

Furthermore, I owe special thanks to many other colleagues from the INNT …

… Kathrin Werner for her laboratory assistance which is greatly appreciated, and without whom I would not have been able to manage the huge amount of work,

… Dr. Martin Eiden and Dipl. Phys. Ariel Viña Rodriguez for the providing the WNV specific qRT-PCRs developed by them,

… Dr. Ute Ziegler and Ines Nedow († 2011) for the introduction in the virus titration assays and the steadily good cooperation, and

… PD Dr. Rainer Ulrich for the constructive suggestions concerning the cell infection experiments.

I also would like to extend my thanks to all my colleagues from the INNT for the good and constructive working atmosphere and for the great team spirit.

I am greatly obliged to my colleagues at the FLI for providing their professional support…

… Mgr. Tomáš Korytář for the countless hours we spent in front of the flow cytometer, his patience, but especially for his enthusiasm and engagement in my work, and his friend-ship,

… Dr. Sasan Fereidouni for his kind offer to construct a phylogenetic tree and for the many discussions on the genetic diversity among WNV strains,

… Dr. Axel Karger for the MALDI-TOF analyses and his numerous advices related to protein isolation and purification,

… Dipl. Chem. Günter Strebelow for the automated RNA extraction of the many hundreds of samples,

… PD Dr. Stefan Finke for the initial training in confocal laser scanning microscopy and his help in establishing a protocol for fluorescence staining,

… Dr. Bernd Köllner and his team for the establishment of a protocol for FACS analysis and for his help with cell transfection,

… Dr. Matthias Lenk and his team from the Cell Bank Department for providing the contin-uous cell lines and culture media,

… Dr. Mario Ziller for the statistical evaluation of the long-term replication study, and

… the animal keepers in the isolation facilities who took care of my mouse lines.

Many external colleagues kindly provided some essential research materials …

… Prof. Kairbaan Hodivala-Dilke, Institute of Cancer, Barts and the London School of Medicine and Dentistry, UK, kindly gifted the β3-deficient mice,

… Dr. Bernhard L. Bader, Technical University Munich - Freising, Germany, provided the the αv-deficient mouse lines,

… PD Dr. Matthias Niedrig, Robert-Koch-Insitute, Berlin, Germany, kindly supplied the WNV New York and Uganda strains,

… Prof. Arno Mullbacher, John Curtis School of Medical Research, Canberra, Australia, made the WNV Sarafend strain available,

… Prof. Dr. Reinhard Faessler, Max Planck Institute, Martinsried, Germany, generously provided the β1-deficient and β1-floxed cells,

… Assoc. Prof. Lieve Naesens, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium, made the MAV-1 available.

I cannot emphasise enough how happy I am to have shared these years and all my experiences with my fellow PhD student and friend Wibke Wohlfromm. We would not have been able to come so far without optimising our daily laboratory routine by team-working and without going through all struggles and challenges together.

I am very grateful to my friends at the Institute Mathias Schlegel, Marc Mertens, Josephine Schlosser, Fabienne Leidel, Imke Dworog and Martin Kaatz for the unforgettable time and fun we had, and for their emotional and their active support in the laboratory I could rely on.

My work profited enormously from their inspirations and ideas, and from having a like-minded platform for discussions and for the exchange of knowledge and experiences.

Last but most importantly I want to give my thanks to my family for their love and encour-agement during those years, to my brother Christian Schmidt for his assistance with computer software, to my mother Edith Schmidt for her unquestionably backing, and to my father Wolfgang Ostendorp not only for his absolute support when the going was though but for his advice and extraordinary help while writing the thesis.

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Im Dokument Cellular factors modulating the entry efficiency of West Nile virus (Seite 169-0)