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Review
. 2015 Nov;2(1):219-39.
doi: 10.1146/annurev-virology-100114-055119. Epub 2015 Jul 2.

Hunting Viral Receptors Using Haploid Cells

Sirika Pillay  1 , Jan E Carette  1
Affiliations
Review

Hunting Viral Receptors Using Haploid Cells

Sirika Pillay et al. Annu Rev Virol. 2015 Nov.

Abstract

Viruses have evolved intricate mechanisms to gain entry into the host cell. Identification of host proteins that serve as viral receptors has enabled insights into virus particle internalization, host and tissue tropism, and viral pathogenesis. In this review we discuss the most commonly employed methods for virus receptor discovery, specifically highlighting the use of forward genetic screens in human haploid cells. The ability to generate true knockout alleles at high saturation provides a sensitive means to study virus-host interactions. To illustrate the power of such haploid genetic screens, we highlight the discovery of the lysosomal proteins NPC1 and LAMP1 as intracellular receptors for Ebola virus and Lassa virus, respectively. From these studies emerges the notion that receptor usage by these viruses is highly dynamic, involving a programmed switch from cell surface receptor to intracellular receptor. Broad application of genetic knockout approaches will chart functional landscapes of receptors and endocytic pathways hijacked by viruses.

Keywords: Ebola virus; Lassa virus; genetic screens; receptor identification; virus entry.

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Figures

Figure 1
Figure 1
Overview depicting the application of haploid cells in forward genetic screening approaches. (a) Mutagenesis of a diploid genome results in heterozygous mutations, which are often phenotypically masked by expression from the remaining wild type allele. In contrast, mutations in haploid cells allow complete genetic knock-out of the gene of interest. (b) In the human haploid genetic screening approaches, retroviral gene trap vectors carrying a splice acceptor site, reporter gene and polyadenylation signal, integrate randomly into the genome of exposed cells. When inserted into intronic (or exonic) regions, the vector disrupts transcription, creating a truncated mRNA transcript, which often results in genetic knock-out. (c) Schematic diagram depicting the steps involved in performing a haploid genetic screen. Haploid cells are randomly mutagenized using a retroviral gene trap vector to create a screening library of high complexity. Cells with mutations in genes that are critical to viral infection are phenotypically selected by infecting with a cytolytic virus of interest. Genomic DNA of the virus-resistant pool is used to PCR amplify DNA flanking the retroviral insertion sites. After deep sequencing and alignment to the human genome, the number of independently generated insertions per gene is counted. These counts are compared to a control dataset derived from cells that are not phenotypically selected. Statistical analysis yields a significance of enrichment value for each gene and these values are plotted to visualize host genes that are deemed essential for virus infection.
Figure 2
Figure 2
Haploid genetic screens reveal viral entry mechanisms via critical intracellular receptors. (a) Ebola virus can be internalized into the endocytic pathway via a number of cell surface receptors. As the endosome matures, resident cathepsin proteases initiate cleavage of a heavily glycosylated domain from the surface glycoprotein of Ebola. Endosome fusion, mediated by the HOPS complex, generates an NPC1 containing endosomal compartment where further cleavage occurs. Interaction of NPC1 then likely triggers fusion of the viral lipid membrane with the host endosomal membrane to allow viral RNA release into the cytosol. (b) Lassa virus is internalized into the endocytic pathway by its cell surface receptor alpha-dystroglycan (DAG1). Acidification of the endosome triggers a receptor switch (dashed arrow) where Lassa virus disengages from alpha-dystroglycan and engages with its intracellular receptor LAMP1. This interaction enables membrane fusion and cytosolic release of viral RNA. Correct dystroglycan glycosylation of DAG1 and N-linked glycosylation of LAMP1 is critical for viral entry as underscored by the near-complete identification of genes in distinct biosynthetic routes of these glycans. Every gene indicated by a gene symbol was found as a significant hit in the screens.
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