Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 238

Fusion is thought to proceed through a hemifusion intermediate in which the outer leaflets of two opposing bilayers fuse, followed by fusion of the inner leaflets and the formation of a fusion pore. Direct evidence for this mechanism has been obtained with influenza virus HA. Mammalian cells in culture producing wild-type HA (left side of figure) are fused with erythrocytes containing two different types of fluorescent dye, one in the cytoplasm (red) and one in the lipid membrane (green). Upon exposure to low pH, HA undergoes conformational changes, the HA1 subunits tilt, and the fusion peptide is inserted into the erythrocyte membrane. The green dye is transferred from the lipid bilayer of the erythrocyte to the bilayer of the HA-producing cell, but the red die is not. Further conformational changes in the HA2 subunits bring the two membranes close together and fusion pores form. As the fusion pores expand, the red dye within the cytoplasm of the erythrocyte is then transferred to the cytoplasm of the HA-producing cell. An altered form of HA (right side of figure) lacking the transmembrane and cytoplasmic domains and with membrane anchoring provided by linkage to a glycosylphosphatidylinositol (GPI) moiety was produced. Upon exposure to low pH, the HA fusion peptide is inserted into the erythrocyte membrane, and green dye is transferred to the membranes of the HA-producing cell, just as in the wild-type protein. However, because no transmembrane domain is present, fusion pores do not form. The diaphragm becomes larger, but there is no mixing of the contents of the cytoplasm, indicating that complete membrane fusion has not occurred. These results prove that hemifusion, or fusion of only the outer leaflet of the bilayer, can occur among whole cells. The findings also demonstrate that the transmembrane domain of the HA protein plays a role in the fusion process.

Glycosylphosphatidylinositol-anchored influenza virus HA induces hemifusion. (Left) Model of the steps of fusion mediated by wild-type HA. (Right) Effect on fusion of an altered form of HA lacking the transmembrane and cytoplasmic domains. Data from Melikyan GB et al. 1995. J Cell Biol 131:679–691.

In contrast to the fusion peptides of class I fusion proteins, fusion loops do not require proteolytic cleavage to be liberated and to be able to insert into membranes. Instead, proteolytic cleavage is required for the conformational change of the second envelope protein, E2 for alphaviruses and prM for flaviviruses (Chapter 13), that shield the fusion loop until the virus particles are delivered in endosomes. Although this cleavage occurs at the Golgi, the process differs for the two virus families. Flavivirus particles bud into the endoplasmic reticulum and are released after passage through the Golgi network, which has a reduced pH. During this process, the E proteins assume the conformation seen on mature particles (Fig. 5.18A). The prM protein is cleaved to pr and M, but the pr fragment continues to shield the fusion loop until the particle is released from the cell, where the pH is neutral. Endocytosis by the target cells returns the virus particles to acid pH, which triggers fusion. On the other hand, alphavirus particles assemble at the plasma membrane and processing of the E2 proteins occurs in the Golgi but prior to their incorporation into particles.

Figure 5.17 SNARE-mediated fusion. The change of syntaxin (t-SNARE, purple) from a closed (not shown) to an open conformation allows SNAP-25 (synaptosome-associated protein of 25 kDa, light and dark orange) and VAMP (vesicle-associated membrane protein, v-SNARE, cyan) helices to “zip up” from their N terminus to their C terminus. The initial “zipping” of the amino-terminal half of SNARE proteins brings the two membranes into nanometer proximity. Completion of assembly of the C termini releases the largest amount of free energy estimated for a protein complex formation and coincides with completion of the fusion process. A number of additional proteins regulate the process (not shown). (PDB ID: 1SCF)

Figure 5.18 Conformational changes in class II proteins during fusion. (A) Ninety dimers of dengue virus envelope glycoprotein E tile the surface of the virus particle. (Inset) Structure of the ectodomains of the dengue protein E dimer is shown at neutral pH (PDB ID: 3J27). Domain I folds into a β-sandwich and is colored orange, domain II cyan/blue, domain III black, and the stem gray. The fusion loop is located at the tip of domain II (red). (B) At low pH, the dimers are disrupted; the proteins extend so that the fusion loop inserts into the target membrane and reorganize into trimers. The glycoprotein then undergoes further conformational rearrangements, folding domain II against domain I, which brings the viral and cell membranes together, allowing fusion. (Inset) Structure of part of the ectodomain of dengue virus E protein at acid pH (based on X-ray crystallographic data; PDB ID: 1OK8), with domains colored as in panel A.