Department of Biosciences and Nutrition Center for Biotechnology Karolinska Institutet, Stockholm, Sweden THE DYNAMIC ENVELOPE OF A FUSION CLASS II VIRUS; MOLECULAR REORGANIZATIONS DURING PREFUSION STAGES OF SEMLIKI FOREST VIRUS

The aim of this study was to explore a membrane fusion mechanism, prevailing in alphaviruses and known as virus class II fusion. The model virus for this mechanism is Semliki Forest virus (SFV). Contrary to class I fusion, for which the influenza virus is the prototype along with HIV, class II fusion mechanism involves membrane proteins mainly folded in -sheet structures, not -helices as in the class I case. The fusion class II viruses enter the cell by fusion with the endosomal membrane. Acidification is a prerequisite for the fusion step to occur and to fulfill the infection in vivo. Virus fusion can be triggered experimentally by acidification in the presence of a target membrane. The acidification would transform the virus into a fusogenic state, after which it is prone to interact with the membrane. Thus, by mimicking the environment in the endosome, stages in the fusion process can be studied under welldefined conditions. In the present work I have focused on the dynamic transformation of SFV at stages preceding membrane fusion. To do so, the accessibility of functional domains in the virus envelope was explored with the aid of antibodies and various biochemical methods. Electron cryo-microscopy (cryo-EM) was used to capture intermediate forms of the virus. This provides data for three-dimensional (3D) structure determination to reveal details and fusion related variations. Pseudo-atomic resolution structures of the virus particle from combined x-ray and cryo-EM data enabled protein assignment to densities within the cryo-EM map. In addition, experiments with formaldehyde (FA) cross-linking of virus particles were performed to gain understanding of its morphological effects. The aim was to develop a method to safely prepare virus specimens for structural studies and, possibly, enhance structural details. In the structures solved, details of acid-induced rearrangements were conclusively identified. The major changes occur as an expansion of the external domain. In the raised shell layer the expanded area show widened shell openings and dissolved protein contacts. In the stalks of the protruding spikes the two glycoproteins separate, but keep together in the spike head lobes, while in the sub-membrane domain the envelope contacts with the nucleocapsid (NC) is released. In spite of the essentially retained virus morphology, several antibody epitopes, including the receptor binding domain and the fusion loop, become exposed in a pH dependent manner. This implies that subtle local rearrangements might represent essential functional stages. In summery, by exploring prefusion stages as they occur in a native virion, this thesis tries to fill a gap in knowledge on viral membrane fusion mechanisms. It shows details of the virus class II fusion mechanism not earlier discussed. It presents new observations and demonstrates sequential stages of rearrangements in the virus structural domains related to the adaptation of a fusogenic state. The results suggest a cooperative action between the two envelope proteins at stages beyond the control of fusion loop exposure. Furthermore, it also presents a procedure to safely prepare virus specimens for structural studies under close to native conditions. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-IV). I. Haag, L. , Garoff, H. , Xing, L. , Hammar, L. , Kan, S. -T. , and Cheng R. H. Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode (2002). EMBO Journal, 21, 4402-4410.2. II. Hammar, L. , Markarian, S. , Haag, L. , Lankinen, H. , Salmi, A. , and Cheng R. H. Prefusion rearrangements resulting in fusion peptide exposure in Semliki Forest virus (2003). Journal of Biological Chemistry, 278, 7189-7198 III. Wu, S. -R. , Haag, L. , Wu, B. , Murata, K. , Xing, L. , Garoff, H. , Hammar, L. , and Cheng, R. H. The dynamic envelope of a fusion type 2 virus; detection of inter and intramolecular reorganizations during prefusion stages. (Manuscript) IV. Haag, L. , Sjöberg, M. , Lindqvist, B. , Garoff, H. , Hammar, L. and Cheng R. H. Formaldehyde inactivated Semliki forest virus show preserved morphological and immunological features. (Manuscript)