Functional Genomic Strategies for Elucidating Human–Virus Interactions

Abstract

Over the last several years a wealth of transformative human-virus interaction discoveries have been produced using loss-of-function functional genomics. These insights have greatly expanded our understanding of how human pathogenic viruses exploit our cells to replicate. Two technologies have been at the forefront of this genetic revolution, RNA interference (RNAi) and random retroviral insertional mutagenesis using haploid cell lines (haploid cell screening), with the former technology largely predominating. Now the cutting edge gene editing of the CRISPR/Cas9 system has also been harnessed for large-scale functional genomics and is poised to possibly displace these earlier methods. Here we compare and contrast these three screening approaches for elucidating host-virus interactions, outline their key strengths and weaknesses including a comparison of an arrayed multiple orthologous RNAi reagent screen to a pooled CRISPR/Cas9 human rhinovirus 14-human cell interaction screen, and recount some notable insights made possible by each. We conclude with a brief perspective on what might lie ahead for the fast evolving field of human-virus functional genomics.

Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics

“…This is consistent with pooled screens using cell survival as a readout displaying limited sensitivity (few candidates recovered) but excellent specificity in finding host genes that act very early in viral replication, for instance host factors needed for viral entry (Perreira et al, 2016). In contrast, RNAi screens generate a larger list of host factor candidates contributing to a broader understanding of viral requirements; however, this comes at the high cost of increased false leads.…”

“…Comparison of candidate lists from similar siRNA screens reveals shared pathways and complexes, but in the majority of instances, there is low exact gene overlap (Perreira et al, , 2016Zhu et al, 2014). The MORR DENV-HF screens behaved similarly, with a low percentage of same-gene overlap detected in the primary screens (for DFs, with % 50% plate mean infected cells and R 50% plate mean number of cells: Silencer Select 13.5%, SMARTpool 14.4%, and SMART-Rev 10.6%; Table S2).…”

CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions

“…The loss of identified host factors blocked key aspects of viral replication and thereby reduced cytotoxicity. Excellent recent reviews describe these analyses [ 43 , 44 ], which will therefore only be covered here briefly. Collectively, the studies outlined below, which we believe to be a comprehensive list of CRISPR-pooled screens of RNA virus biology at the time of this publication, highlight the significant potential for CRISPR loss-of-function analysis for studies of host dependency factors.…”

“…CRISPR–Cas9 screens independently identified endoplasmic reticulum (ER) pathways important for flavivirus replication. These include screens of West Nile [ 31 , 45 ], dengue [ 44 ], Zika [ 46 ], and hepatitis C virus [ 47 ] replication, which used the rescue from viral cytopathic effect as the method of positive selection. The N-linked glycosylation pathway, ER-associated degradation, and ER signal peptide pathway strongly scored in these flavivirus replication screens.…”

“…Intracellular adhesion molecule 1 (ICAM1), the known HRV receptor, was one of the two high-confidence hits. The exocyst-targeting and vesicular transport pathway component Exocyst Complex Component 4 (EXOC4) was the other hit, whose role in supporting HRV infection remains to be further characterized [ 44 ]. A CRISPR–Cas9 screen for T cell factors important for C-C motif chemokine receptor 5 (CCR5)-tropic human immunodeficiency virus (HIV) infection identified the obligatory receptors CD4 and CCR5.…”