are shown, four of which are essential for entry. Initial attachment to HSPG (heparan sulfate glycosaminoglycans) is mediated by gC and gB. gD engages the main receptor, which can be either nectin-1, HVEM (herpesvirus entry mediator), or 3-OS-HS (3-O-sulfated heparan sulfate). Subsequently, gD binds the gH/gI heterodimer, which then activates gB to mediate fusion. The entry route can differ depending on the cell. For example, the gK protein enables fusion at the plasma membrane of neurons.
Entry into Cells
Following attachment to one or more receptors, virus particles have to enter the cells. Many animal viruses enter cells by the same pathways by which cells take up macromolecules. The plasma membrane, the limiting membrane of the cell, permits nutrient molecules to enter and waste molecules to leave, thereby ensuring an appropriate internal environment. Water, gases, and small hydrophobic molecules such as ethanol can freely traverse the lipid bilayer, but most metabolites and ions cannot. These essential components enter the cell by multiple transport processes (Fig. 5.10). Obviously, receptors play a role is this process, as they can localize at specific membrane domains prior to or after virus particle attachment and can also mediate signaling that facilitates virus particle up-take by processes normally employed to allow molecules to enter the cell. Disruption of cellular membranes is a necessary step in virus entry and distinguishes enveloped from nonenveloped viruses. For the former, membrane fusion is an integral step of entry, whereas nonenveloped viruses use alternative mechanisms described in later sections. Typically, entry and intracellular transport are tightly linked, though this section will focus on how viruses enter cells.
Virus-Induced Signaling via Cell Receptors
Binding of virus particles to cell receptors not only concentrates the particles on the cell surface, but also activates signaling pathways that facilitate virus entry and movement within the cell or produce cellular responses that enhance virus propagation and/or affect pathogenesis. Binding of virus particles may lead to activation of protein kinases that trigger cascades of responses at the plasma membrane, cytoplasm, and nucleus (Chapter 14). Second messengers that participate in signaling include phosphatidylinositides, diacylglycerides, and calcium. Regulators of membrane trafficking and actin dynamics also contribute to signaling. Additionally, virus-receptor interactions can stimulate antiviral responses, such as synthesis of type I interferons (Volume II, Chapter 3).
Figure 5.10 Mechanisms for the uptake of macromolecules from extracellular fluid. During phagocytosis, large particles such as bacteria or cell fragments that come in contact with the cell surface are engulfed by extensions of the plasma membrane. Phagosomes ultimately fuse with lysosomes, resulting in degradation of the material within the vesicle. Endocytosis comprises the invagination and pinching off of small regions of the plasma membrane either by macropinocytosis or by receptor-mediated endocytosis. Macropinocytosis mediates nonspecific uptake of fluids and small molecules. It is triggered by ligands and dependent on actin and signaling pathways different from those required for phagocytosis. Receptor-mediated endocytosis results in the specific uptake of molecules bound to cell surface receptors. Filopodia are actin-rich protrusions that sample the environment and participate in many cellular processes such as migration. Movement along filopodia occurs by an actin-dependent mechanism.
Signaling is essential for the entry of some viruses, such as simian virus 40, into cells. Binding of this virus particle to its glycolipid cell receptor, GM1 ganglioside, causes activation of tyrosine kinases. The signaling that ensues induces reorganization of actin filaments, internalization of the virus in caveolae, and transport of the caveolar vesicles to the endoplasmic reticulum (Fig. 5.6). The activities of more than 50 cellular protein kinases regulate the entry of this virus into cells.
Routes of Entry
A wide range of ligands, fluid, membrane proteins, and lipids are taken into cells from the extracellular milieu by various processes depending on their size (Fig. 5.10). Phagocytosis and endocytosis mediate the uptake of larger and smaller, respectively, molecules and particles. Endocytosis is the mechanism of entry of many viruses (Fig. 5.10 and 5.11). Three pathways of endocytosis have been identified: clathrin-dependent, caveolin-dependent, and clathrin- and caveolin-independent. Clathrin-coated pits can comprise as much as 2% of the surface area of a cell, and some receptors are clustered therein even in the absence of their ligands, whereas others require ligand binding to cluster. Successive transport and fusion with late endosomes exposes the contents of the vesicles to increasingly acidic pH—6.5 to 6.0 (early) and 6.0 to 5.0 (late)—while in lysosomes they are exposed to multiple degradative enzymes. Viral fusion proteins with a high pH threshold for fusion, such as that of vesicular stomatitis virus, mediate entry from early endosomes; but most mediate entry into the cytoplasm from late endosomes, and a few from lysosomes.
The caveolin-dependent pathway participates in transcytosis, signal transduction, and uptake of membrane components and extracellular ligands. Binding of a virus particle to the cell surface activates signal transduction pathways required for pinching off caveolae, which then are transported within the cytoplasm. These vesicles ultimately fuse with the caveosome, a larger membranous organelle that contains caveolin (Fig. 5.11). In contrast to endosomes, the pH of the caveosome lumen is neutral. Some viruses, like echovirus type 1, penetrate the cytoplasm from the caveosome.
Although in cell culture virus particles can enter cells preferentially by one pathway, many viruses appear indiscriminate and enter via multiple pathways. For example, herpes simplex virus can enter cells by three different routes and simian virus 40 is taken up by both caveolin-dependent and clathrin-/caveolin-independent pathways. Influenza A virus can enter cells by both clathrin-dependent endocytosis and macropinocytosis, a process by which extracellular fluid is taken into cells via large vacuoles. Many virus particles are taken up by this pathway, including vaccinia virus, ebolaviruses, and herpesviruses (even though the latter can also fuse at the plasma membrane). Upon receptor binding, viruses that enter cells via macropinocytosis trigger a signaling cascade that leads to changes in cortical actin and ruffling of the plasma membrane (Fig. 5.10). When these plasma membrane extensions retract, the viruses are brought into macropinosomes and eventually leave these vesicles via membrane fusion. A more dramatic rearrangement of actin filaments leads to the formation of filopodia, thin extensions of the plasma membrane. Virus particles of polyomaviruses can be visualized moving laterally on the plasma membrane on filopodia, and filopodia bridges participate in cell-to-cell spread of retrovirus particles in cells in culture (Chapter 13).
Figure 5.11 Virus entry and movement in the cytoplasm. Examples of various routes of virus entry are shown. Fusion at the plasma membrane releases the nucleocapsid or subviral components (lower left side of the cell) that can travel on actin filaments and microtubules. Uptake of virus particles by endocytosis can be either clathrin dependent, caveolin dependent, or clathrin
