Genome Screen Identifies Host Proteins Required for Flavivirus Infection

A team of molecular virologists employed advanced genome editing techniques to identify the host proteins required by flaviviruses such as dengue and Zika for infection and replication.

Investigators at the University of Massachusetts Medical School (Worcester, USA) applied CRISPR/Cas9 and RNAi techniques to screen for proteins required by the dengue and Zika viruses.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas complex comprises a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR-Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides (CRISPRs) that shepherd the Cas9 protein to the target gene on a DNA strand.

The RNAi (interfering RNA) approach depends on microRNAs (miRNAs), which are a small noncoding family of 19- to 25-nucleotide RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, inducing translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and their targets.

The investigators reported in the June 21, 2016, online edition of the journal Cell Reports that their screen of host proteins had identified the Zika virus entry factor AXL (receptor tyrosine kinase) as well as multiple Ras-related protein host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). Both flaviviruses required the EMC for their early stages of infection.

"These genetic screens give us our first look at what these viruses need to survive," said senior author Dr. Abraham Brass, assistant professor of microbiology and physiological systems at the University of Massachusetts Medical School. "Our lab and others in our field have worked hard to develop the systems and infrastructure needed to investigate the genetics underlying how viral pathogens use our own cell's machinery to replicate. This has allowed the scientific community to respond quickly when the Zika virus threat emerged. In our lab, we adapted the technology and tools we had established over the last four years working with other viruses to begin investigating the biology of Zika virus. We plugged Zika virus into our system and immediately began studying it. What might have taken much longer to build from the ground up, we were able to turn around in a few short months. Our goal was to get the screens done, find what the viruses need to grow, and then get the data out to the rest of the research community right away."

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