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Class I viral fusion glycoproteins facilitate fusion of the viral envelope with cell membranes and entry of the virus into the cell, through extensive short sequence-specific interactions. Regions mediating these interactions include the N-terminal hydrophobic fusion peptide, a pair of extended 4,3hydrophobic heptad repeats (HRs), a membrane-active membrane proximal external region (MPER), a hydrophobic transmembrane domain and the cytoplasmic tail region. In particular, the anti-parallel binding of the C-terminal HR to the central N-HR trimeric coiled-coil forms the 6-helix bundle fusion core. These interaction-mediating sequences are generally well preserved sequentially and structurally, allowing their peptidyl analogues to be developed as antiviral therapeutics and/or research reagents (e.g. HR-derived peptides). Novel targets for the development of antiviral drugs and viral detection reagents are required when facing drug-resistant viral strains, viral pathogens without effective and/or economical treatment, and newly emerging viral pathogens. This thesis focuses on the systematic identification of novel interaction-mediating sequences within Class I viral fusion glycoproteins, and the investigation of their involvements in viral replication as well as their potential applications in diagnosis and anti-viral interventions. In Paper I, peptide array scanning identified 34 spike (S) protein-derived peptides that bound to the S protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). These putative self-binding peptides contain five core octapeptide consensus sequences, among which the octapeptide GINITNFR was predicted to form β-zipper-mediated amyloid-like fibrils. The peptide C6 containing this sequence was subsequently shown to oligomerize and form amyloid-like fibrils. The potential of C6 to conduct β-zipper-mediated interactions was further applied to detect the S protein expression by immunofluorescence staining. The peptide array scanning in Paper I used the S protein ectodomain without the MPER and beyond. Using chemical crosslinking and immunofluorescence staining, in Paper II we could show that the S protein MPER could oligomerize and further heteromerize with the N-terminal internal fusion peptide (IFP). The MPER-derived peptides also inhibited the coronavirus entry in a dose-dependent manner, potentially through disrupting the MPER-mediated interactions. The ability of peptides derived from the MPER in inhibiting viral entry and infection was subsequently studied in Paper III, in the context of HIV-1. The antiviral activities of the HIV-1 Env MPER-derived peptides were abrogated upon Ala substitution of the Trp residues or deletion of the Cterminal cholesterol-interacting motif. Unexpectedly, Ala substitutions of the Trp residues within HIV-1 Env significantly elevated the biosynthesis of another viral structural protein, the p55/Gag, which led to enhanced viral particle release. In Paper IV, besides the MPER we identified the signal sequence of HIV-1 Env as another region that could negatively regulate the expression of p55/Gag. The HIV-1 Env signal sequence, which mediates the co-translational translocalization of nascent Env polypeptide into the endoplasmic reticulum, inhibited the viral protein expression and production, probably at a post-ER-targeting stage. N-terminal truncations of the Env signal sequence significantly elevated the intracellular and intraviral levels of late viral proteins and the proviral genome transcription in a timeand dose-dependent fashion. Moreover, the truncations suppressed the HIV-1 promotor (LTR)-driven expression of a reporter protein, suggesting that the Env signal sequence inhibited viral genome transcription through LTR-dependent interactions. This thesis focused on three novel interaction-mediating sequences within two Class I viral fusion glycoproteins, which could regulate the viral infectivity, at both viral entry and assembly, through protein-protein, protein-lipid, and/or protein-nucleic acids interactions. These sequences and the interactions that they are meditating could be further targeted by their peptidyl analogues for viral detection and/or inhibition. LIST OF SCIENTIFIC PAPERS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals (I-IV). I. Si Min Zhang, Ying Liao, Tuan Ling Neo, Yanning Lu, Ding Xiang Liu, Anders Vahlne, James P. Tam. Identification and applications of selfbinding zipper-like sequences in SARS-CoV spike protein. Manuscript. II. Ying Liao, Si Min Zhang, Tuan Ling Neo, James P. Tam. Tryptophandependent membrane interaction and heteromerization with internal fusion peptide by membrane proximal external region of SARS-CoV spike protein. Biochemistry. 2015; 54 (9): 1819−1830. These authors contributed equally. III. Si Min Zhang, Alenka Jejcic, James P. Tam, Anders Vahlne. Membraneactive Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins. PLoS ONE. 2015; 10(7): e0134851. IV. Si Min Zhang, Anders Vahlne, Alenka Jejcic. The gp160 Signal Sequence Suppresses HIV-1 Replication in an LTR-Dependent Manner. Manuscript. CONTENTS

Interaction-mediating sequences within class I viral fusion glycoproteins : their roles in viral infection and in applications