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Introduction – infection cycle

_________________________________________________________________________ Cell entry of enveloped viruses Steven AC and Spear PG. Science VOL 313, 2006 Binding of viral glycoprotein with cell surface receptor lead to the fusion of viral envelope with the plasma membrane Binding of viral glycoprotein with cell surface receptor triggers receptor mediated endocytosis (receptor/clathrin/caveolae)

_________________________________________________________________________ Cell entry of enveloped viruses Nature Reviews Microbiology 6, (February 2008) low-pH mediated fusion receptor-triggered fusion

_________________________________________________________________________ Viral glycoprotein structure Viral surface glycoproteins essential for virus-cell membrane fusion (e.g. HIV (gp41/gp120) & Influenza HA1/HA2) structure of glycoprotein: - external domain (host interaction) - endodomain (internal part) - transmembrane domain (spans the viral envelope membrane; typically  -helix) Nature Reviews Microbiology 6, (February 2008)

_________________________________________________________________________ Classes of virus fusion proteins Class I Class II Class III glycoprotein B of herpes simplex virus (HSV) gBgB glycoprotein G of vesicular stomatitis virus (VSV)

_________________________________________________________________________ Classes of virus fusion proteins priming step of fusion proteins during budding process: - proteolysis of precusrsor (e.g. HA0) or co-regulatory protein (e.g. p62) Trigger mechanisms (low pH or co-receptor binding) induce conformational change of fusion protein to a extended intermediate due to altered energy profile

_________________________________________________________________________ Main mechanism of membrane fusion Nature Reviews Microbiology 6, (February 2008); Nat Struct Mol Biol July, 15(7): 690–698.

_________________________________________________________________________ Class I membrane-fusion proteins (Influenza) cell virus Nat Struct Mol Biol July, 15(7): 690–698 Metastable HA2 protein trimer with transmembrane domain, α-helical coiled coil stalk and buried fusion peptide conformational change and HA1 dissociation upon specific trigger mechanism (HIV -> CD4 receptor; Influenza -> sialic acid & low pH) Loop-to-helix transition, translocation of fusion peptide towards cell membrane and anchoring -> pre-hairpin intermediate (high energy) followed by hemifusion Folding-back bridge collapsing to stable α-helical coiled coil hairpin-trimer and fusion peptide capping of transmembrane domain -> irreversible hairpin fusion pore

_________________________________________________________________________ Class II membrane-fusion proteins (flavivirus and alphavirus) Nat Struct Mol Biol July, 15(7): 690–698 folding with regulatory companion protein: flavivirus (p62); alphavirus (prM) β-sheet (red), fusion loop domain (yellow) and Ig-like (blue) domain connected with TM in metastable form: flavivirus (E) 2 homodimer; alphavirus (E1-E2) 3 heterodimer fusion peptides

_________________________________________________________________________ Class II membrane-fusion proteins (flavivirus) Nat Struct Mol Biol July, 15(7): 690–698 Metastable E protein homodimer (alphavirus: heterodimer) linked to transmembrane domain, buried fusion peptide (black circle) Dimer dissociation upon specific trigger mechanism (H + binding at low pH) and trimerization to homotrimers upon fusion peptide contact to target membrane Extended intermediate and fusion peptide anchoring upon interaction of domains I and II -> pre-hairpin intermediate (high energy) followed by hemifusion Flipping-over (arrows) of domain III lead to back-zipping of stem, bringing fusion loop and transmembrane domain together -> irreversible hairpin fusion pore cell virus

_________________________________________________________________________ Class II membrane-fusion proteins (alphavirus)

_________________________________________________________________________ Class III membrane-fusion proteins (VSV) Nat Struct Mol Biol July, 15(7): 690–698 G-protein fusion trimer with core domain (red) and fusion peptide (black circle) held away from target membrane Flipping over of fusion domain (arrow) upon specific trigger mechanism (pH) and fusion peptide contact to target membrane -> extended intermediate Post fusion conformation of the subunit (d) and the trimer (e) after back-folding and up-zipping of transmembrane domain bringing the membranes together cell virus

_________________________________________________________________________ Important factors that contribute to virus membrane fusion cholesterol and sphingolipid content of target cell membranes cooperative interaction of fusion peptides to cluster rings of five or six protein homotrimers -> enhancement of initial fusion pore formation (´dome like´) requirement of trimers for membran fusion: - Influnenza -> HIV -> 1 !!

_________________________________________________________________________ Summary I Dev Cell Jan;14(1): Review.

_________________________________________________________________________ Summary II Modified from Nat Rev Microbiol Jan;4(1): Review. Class III (VSV G) reversible trimer formation upon pH change α-helix + β-sheet No proteolytic process No companion Internal loop at fusion protein Trimer of hairpins

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_________________________________________________________________________ Classes of virus fusion proteins Class I Found in othomyxoviruses, paramyxoviruses, retrovoruses, filoviruses and coronaviruses. Form trimers with triple coiled-coil stem which in the postfusion state gives a distinctive six-helix bundle. The active fusogenic form is obtained through a proteolytic cleavage which reveals the hydrophobic fusion peptide. Class II Found in flaviviruses and alphaviruses Initially are present in form of dimers but at low pH the dimers dissociate exposing the fusion loops. The fusion loops insert into host membrane which triggers trimerization of the glycoproteins. Class III Glycoprotein B of Herpes Simplex Virus (HSV) gBgB Glycoprotein G of Vesicular Stomatitis Virus (VSV)

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