A Whole-Genome RNA Interference Screen for Human Cell Factors Affecting Myxoma Virus Replication

Abstract

Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro-and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to ␤-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G 1 , or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G 1 /S boundary with an inhibitor of cyclindependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection

“…GFP signals were normalized to cell number values, and control experiments were set to 100% infection. For this primary screen, hit selection was based on Z-score statistics; this method was used because the large number of siRNAs typically produces a frequency distribution with an approximate Gaussian curve, albeit with a slight skew ( 38 ). Using a Z-score threshold set at −3, or 3 standard deviations (SDs) from the mean, we restricted our genes of interest to only those that decreased RVFV MP12-GFP infection and identified 2,053 gene targets.…”

Stress Beyond Translation: Poxviruses and More

“…Heat shock proteins play critical roles for cells, especially under stress conditions. Interestingly, heat shock responsive pathways are also particularly important for poxvirus infection [ 13 , 14 , 15 , 16 ]. How poxviruses manipulate heat shock response for their own benefit is an interesting subject.…”

Identification of host DEAD-box RNA helicases that regulate cellular tropism of oncolytic Myxoma virus in human cancer cells

“…By pharmacological manipulation of AKT phosphorylation, it was possible to significantly increase MYXV replication in some semipermissive/nonpermissive human cancer cells 31 . In an effort to identify additional host factors that either restrict or enhance MYXV replication in human cancer cells, a genome-wide small interfering RNA (siRNA) library screen has been performed 32 . This screen has reported identification of large set of genes that can either enhance or reduce MYXV replication in human MDA-MB231 breast cancer cells 32 .…”