and caveolin independent. Clathrin-dependent endocytosis (top left side of the cell) commences with binding to a specific cell surface receptor followed by diffusion into an invagination of the plasma membrane coated with the protein clathrin on the cytosolic side (clathrin-coated pits). The coated pit further invaginates and pinches off, a process that is facilitated by the GTPase dynamin. Within a few seconds, the clathrin coat is lost and the vesicles fuse with small, smooth-walled vesicles located near the cell surface, called early endosomes. The lumen of early endosomes is mildly acidic (pH 6.5 to 6.0), a result of energy-dependent transport of protons into the interior of the vesicles by a membrane proton pump. The contents of the early endosome are then transported via endosomal carrier vesicles to late endosomes located closer to the nucleus. The lumen of late endosomes is more acidic (pH 6.0 to 5.0). Late endosomes in turn fuse with lysosomes, which are vesicles containing a variety of enzymes that degrade sugars, proteins, nucleic acids, and lipids. Particle uncoating usually occurs from early or late endosomes. Virus particles may enter cells by a dynamin- and caveolin-dependent endocytic pathway (right side of the cell). Caveolae are distinguished from clathrin-coated vesicles by their flask-like shape, their smaller size, the absence of a clathrin coat, and the presence of a marker protein called caveolin. Three types of caveolar endocytosis have been identified. Dynamin 2-dependent endocytosis by caveolin 1-containing caveolae is observed in cells infected with simian virus 40 and polyomavirus. Dynamin 2-dependent, noncaveolar, lipid raft-mediated endocytosis occurs during echovirus and rotavirus infection, while dynamin-independent, noncaveolar, raft-mediated endocytosis is also observed during simian virus 40 and polyomavirus infection. This pathway brings virions to the endoplasmic reticulum via the caveosome, a pH-neutral compartment. Clathrin- and caveolin-independent endocytic pathways of viral entry have also been described (top center-right of cell). Movement of endocytic vesicles within cells occurs on actin filaments or microtubules, the components of the cytoskeleton. Actin filaments are two-stranded helical polymers of actin. They are dispersed throughout the cell but are most highly concentrated beneath the plasma membrane, where they are connected via integrins and other proteins to the extra-cellular matrix. Transport along actin filaments is accomplished by myosin motors. Microtubules are 25-nm hollow cylinders made of tubulin. They radiate from the centrosome to the cell periphery. Movement on microtubules is mediated by kinesin and dynein motors.
Membrane Fusion
In the case of enveloped viruses, fusion between viral and cellular membranes must occur to deliver the viral nucleic acid into the cell. Membrane fusion takes place during many cellular processes, such as cell division, myoblast fusion, and exocytosis, and must be regulated in order to maintain the integrity of the cell and its intracellular compartments. Consequently, membrane fusion proceeds by specialized mechanisms mediated by proteins and requires energy. Some of the best-characterized fusion machines are viral envelope glycoproteins.
Envelope glycoproteins from different virus families appear utterly dissimilar in primary amino acid sequence and domain organization, structure, and even function. Receptor binding and fusion of some virus particles are mediated by the same protein. For others, these functions are segregated into two distinct proteins. Some viral proteins can mediate fusion at the cell surface, while others require activation by acidic pH in endocytic vesicles. Nevertheless, despite these differences, the fusion mechanism is remarkably similar between all fusion proteins from different virus families and relies on conformational changes in the viral protein or subunit that mediates fusion. Based on protein structure, viral fusion proteins can be assigned to one of three classes. Class I includes the most extensively studied examples of fusion proteins, exemplified by influenza virus HA. The study of this protein elucidated the mechanism of fusion with general features that are common to all fusion proteins regardless of class.
Class I Fusion Proteins
In addition to influenza virus HA, this class includes the human immunodeficiency virus type 1 envelope glycoprotein and paramyxovirus fusion proteins. These proteins are initially synthesized as a polyprotein precursor that is thermo-dynamically stable until subsequently cleaved. Proteolytic cleavage is a determinant of tropism: for example, inefficient cleavage of some avian influenza HA protein precursors in mammalian cells limits their zoonotic potential. This tropism restriction occurs because cleavage is essential for the release of the fusion peptide, a highly hydrophobic sequence that can insert into lipid membranes and that lies at the cleaved, extreme N terminus of the transmembrane subunit. Following cleavage, the fusion peptide has to be sequestered until the virus particle leaves the producing cell and reaches the target cellular membranes; otherwise it can insert into membranes prematurely.
The process is best illustrated for influenza virus HA. In the envelope protein precursor, HA0, the fusion peptide sequence is positioned at the junction of the two subunits HA1 and HA2, forming a loop where proteolytic cleavage occurs (Fig. 5.12). After cleavage, the fusion peptide translocates to a cavity formed by both HA1 and HA2 residues so that its hydrophobic residues are buried. In order for fusion to occur, the fusion peptide must be exposed. Therefore, protein regions that keep the fusion peptide shielded have to rearrange. This rearrangement is dependent on a signal indicating that the virus particle has reached the appropriate target membrane and is known as the fusion trigger. Acid pH is a common fusion trigger for viral fusion proteins, including those from class II and III, but fusion triggers can differ among viruses. In all cases, however, following exposure, the fusion peptide must reach the target membrane in order to penetrate it. This movement is achieved by extensive conformational changes in the transmembrane subunits of fusion proteins (Fig. 5.12). Upon fusion activation, protein segments that previously held the fusion peptide close to the transmembrane subunit core structure fold to project the fusion peptide toward the target membrane.
Figure 5.12 Conformational changes of class I proteins during fusion. The envelope glycoprotein of influenza viruses is synthesized as a precursor, HA0, that is cleaved at a specific sequence that forms a loop (green and red). For simplicity, the diagram of only one of three members of a trimer is shown, with HA1 in gray and HA2 in various colors to highlight conformational changes (PDB ID: 1HA0, 4FNK, 1HTM). The viral membrane would be located at the bottom of the image and the target membrane at the top. Following cleavage, HA1 and HA2 remain attached by disulfide bonds and other interactions. The fusion peptide (red) is now buried in the stem of the trimer structure, but the overall structure of the protein is not altered. HA1 mediates binding to the receptor and virus particles are endocytosed. Import of protons into the endosome triggers conformational rearrangements. The HA1 subunits tilt away from the core to expose the fusion peptide (not shown). A segment of HA2 (black) assumes a helical conformation that now extends N-terminal sequences, including the fusion peptide, toward the target membrane. At the C terminus of the protein, segments (blue and purple) fold against the central helix (cyan). This fold, known as the hairpin, brings the viral membrane at the C terminus of HA2 close to the target cell membrane so that fusion can occur.
Insertion of the fusion peptide into the target membrane results in the adoption of an extended intermediate structure. This structure bridges the two membranes but does not bring them close enough for fusion to occur (Fig. 5.13). Consequently, additional changes in protein conformation are required. Amino acid sequence comparison reveals a second region, in addition to the fusion peptide, that is similar in several types of viral fusion proteins. This region is a heptad repeat (a repeated sequence of seven amino acids) consisting of leucine or isoleucine residues that folds into an α-helical coiled coil known as a leucine zipper. Leucine zippers mediate protein-protein interactions and were therefore originally thought to be responsible for envelope protein oligomerization.
