The primary cell receptor for human immunodeficiency virus type 1 is CD4, a 55-kDa rod-like protein that is a member of the Ig superfamily and has four Ig-like domains (Fig. 5.3). A variety of techniques have been used to identify the site of interaction with human immunodeficiency virus type 1, including site-directed mutagenesis and X-ray crystallographic studies of CD4 bound to the viral attachment subunit SU (Fig. 5.8B). The CD4-binding site in SU is a deep cavity, and the opening of this cavity is occupied by CD4 amino acid Phe43, which is critical for SU binding. This Phe43 is in a region analogous to a Phe127 in CD155 that binds to poliovirus. Remarkably, two viruses with entirely different architectures bind to analogous surfaces of these Ig-like domains. Comparison of the structure of SU in the presence and absence of CD4 indicates that receptor binding induces conformational changes in SU. These changes expose binding sites on SU for the chemokine receptors, which are required for fusion of viral and cellular membranes (see Box. 5.1 and “Membrane Fusion” below).
Figure 5.8 Interaction of human immunodeficiency virus type 1 envelope glycoprotein with its receptor. (A) The HIV-1 envelope precursor is cleaved to produce two subunits, SU (surface) and TM (transmembrane), that remain noncovalently attached. (B) Ribbon diagram of an SU monomer (gray) bound to CD4 (purple) (based on X-ray crystallographic data; PDB ID: 2NY1). SU residues that form the CD4-binding site are colored green. The side chain of CD4 Phe43, a residue critical for binding to SU, is shown penetrating the hydrophobic cavity of SU. This amino acid, which makes 23% of the interatomic contacts between CD4 and SU, is at the center of the interface and appears to stabilize the entire complex.
Virus particles with multiple attachment and entry proteins. Entry appears far more complicated in the case of herpesviruses, which have multiple surface glycoproteins that coordinate in different ways to guide entry via distinct routes depending on the target cell. The components affecting selection of particular routes of entry are not well understood. For example, herpes simplex virus 1 particles have 12 glycoproteins and 3 nonglycosylated proteins on their surface. Initial contact of virus particles with the cell surface is made by low-affinity binding of two viral glycoproteins, gC and gB, to glycosaminoglycans (preferentially heparan sulfate), abundant components of the extracellular matrix (Fig. 5.9). Such interactions concentrate virus particles near the cell surface and facilitate subsequent attachment of the viral glycoprotein gD to one of its three receptors. Members of at least two different integral membrane protein families serve as entry receptors for alphaherpesviruses: nectin-1, a member of the nectin family that includes the poliovirus receptor CD155 (yet another example of receptors shared by different viruses); and herpesvirus entry mediator, a member of the tumor necrosis receptor family. However, when members of these two protein families are not present, gD can engage 3-O-sulfated heparan sulfate as a receptor for viral entry. Receptor binding induces conformational changes in gD that allow it to bind to the gH/gL heterodimer, which in turn activates gB to mediate fusion. gD, gH, gL, and gB are the only glycoproteins required for entry, and their interactions with cellular proteins can drive selection of a particular entry route. However, other herpes simplex virus 1 envelope proteins can also influence this selection (Fig. 5.9).
Cell Surface Lectins and Spread of Infection
Virus particle attachment to certain cell surface proteins may not mediate entry into that particular cell but might facilitate dissemination within a host. An example is the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), a tetrameric lectin present on the surface of dendritic cells (Fig. 5.3). This lectin binds high-mannose, N-linked glycans, such as those produced in insect cells. Viruses that reproduce in insects are delivered to the human skin via a bite and may bind and sometimes infect dendritic cells. These cells then carry the viruses to other parts of the body, particularly lymph nodes. However, not all viruses that bind DC-SIGN replicate in insect cells. In humans, DC-SIGN on the surface of dendritic cells binds human immunodeficiency virus type 1 virus particles, but cell entry does not take place. In cells in culture, dendritic cells can store and release infectious virus. Therefore, while the interaction of human immunodeficiency virus type 1 with DC-SIGN is nonproductive, it may lead to viral dissemination in the host when dendritic cells migrate to lymph nodes rich in the virus target, CD4+ T cells (see Chapter 13).
Figure 5.9 Multiple receptors for herpes simplex virus 1 (HSV-1). Six (of 15) viral surface glycoproteins 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