Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 223

Decades of technological advances have enabled the identification of many receptors for viruses. First, production of monoclonal antibodies provided a powerful means of isolating and characterizing individual cell surface proteins. Hybridoma cell lines that secrete monoclonal antibodies that block virus attachment are obtained after immunizing mice with intact cells. Such antibodies can be used to purify the receptor protein by affinity chromatography.

A second technology that facilitated the identification of receptors was the development of cell transfection with DNA. This method was crucial for isolating genes that encode receptors, following introduction of DNA from susceptible cells into nonsusceptible, but otherwise permissive, cells (Fig. 5.2). Cells that acquire DNA encoding the receptor and carry the corresponding protein on their surface are able to bind virus particles specifically. Clones of such cells are recognized and selected by several different methods (Fig. 5.2). The receptor genes can then be isolated from these selected cells using molecular cloning. These technologies were groundbreaking when first developed and continue to be very useful. For example, the receptor for Middle Eastern respiratory syndrome coronavirus was identified just 4 months after the first description of the virus using affinity isolation with a hybrid envelope-antibody molecule.

As new technologies become available, they are also employed in the identification of viral receptors. The murine norovirus and the bat influenza A virus receptors were identified using a genome-wide CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 screen (Chapter 2). After multiple rounds of infection, single guide RNAs targeting plasma membrane proteins were enriched in cells that survived, demonstrating that these proteins were important for infection by the virus used in the screen. Screens that rely on knocking down gene expression benefit from the availability of haploid cells, which carry a single copy of each gene. A human haploid cell line proved very useful in the identification of ebolavirus and Lassa virus receptors by insertional mutagenesis. As with every screen, the function of each protein identified as a viral receptor was confirmed using additional experimental approaches.

Figure 5.2 Experimental strategies for identification and isolation of genes encoding cell receptors for viruses. (A) Genomic DNA or pools of DNA clones made from messenger RNA from cells known to synthesize the receptor are introduced into receptor-negative permissive cells. When complementary DNAs (cDNAs) cloned in a plasmid are used as the donor DNA, pools of individual clones (typically thousands) are prepared and introduced into cells. A small number of transduced cells will produce the receptor. The specific DNA pool that yields receptor-producing cells is then subdivided, and the screening process is repeated until a single receptor-encoding DNA is identified. Three different strategies for identifying such rare receptor-expressing cells are shown. (I) The cells are infected with a virus with a genome that has been engineered to carry a gene encoding drug resistance. Cells that express the receptor will become resistant to the drug. This strategy works only for viruses that persist in cells without killing them. (II) For lytic viruses, an alternative is to engineer the viral genome to express an indicator, such as green fluorescent protein or β-galactosidase. Cells that make the correct receptor and become infected with such viruses can be distinguished by the apparent fluorescent color change. (III) The third approach depends on the availability of an antibody directed against the receptor, which binds to cells that express the receptor gene. Bound antibodies can be detected by an indicator molecule. (B) Alternatively, approaches that rely on survival of receptor-negative cells can be employed. In such strategies, genome-wide screens are performed by either CRISPR/Cas9 or alternative methods that aim to disrupt receptor expression in susceptible and permissive cells. Cells are then infected with a cytopathic virus or a recombinant cytopathic virus, such as vesicular stomatitis virus, carrying the envelope protein of the virus under investigation. Disrupted genes can then be identified in surviving cells. In all strategies, iterative rounds of screening are performed.

The availability of receptor genes has made it possible to investigate the details of receptor interaction with virus particles by site-directed mutagenesis. Receptor proteins can be synthesized in heterologous systems and purified, and their properties can be studied in vitro, while cells in culture that produce altered receptor proteins can be used to test how they affect virus attachment. Because of their hydrophobic membrane-spanning domains, many of these cell surface proteins are relatively insoluble and difficult to work with. Although recent advances in cryo-electron microscopy can now aid in determining the structure of large complexes and transmembrane proteins, soluble extracellular protein domains (that contain the virus binding sites) have been essential for structural studies of receptor-virus interactions.

Receptor genes have also been used to produce transgenic mice that synthesize receptor proteins. Such transgenic animals can serve as useful models in the study of human viral diseases. For example, mouse cells are permissive for poliovirus reproduction, and susceptibility is limited only by the absence of the virus receptor. Consequently, it was possible to develop a small-animal model for poliomyelitis by producing transgenic mice that synthesize this receptor. Inoculation of these transgenic mice with poliovirus by various routes produces paralysis, as is observed in human poliomyelitis. These mice were the first new animal model created by transgenic technology for the study of viral disease. Similar approaches have subsequently led to animal models for diseases caused by measles virus and echoviruses.