Abstract
Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2aPol levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2aPol localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.
Highlights
Viruses survive with limited genetic material by interacting with and exploiting host factors at essentially every replication step [1,2,3,4,5,6,7,8,9,10,11,12,13,14]
We have analyzed,93% of all yeast genes (,5,800), and the 123 host genes identified to date that affect Brome mosaic virus (BMV) RNA replication represent 2.3% of the yeast genome
Our studies to date likely underestimate the number of host genes that contribute to BMV RNA replication. ,60% of non-essential yeast genes are genetically redundant, meaning that the functions of many gene deletions are partially compensated for by other genes [39,40]
Summary
Viruses survive with limited genetic material by interacting with and exploiting host factors at essentially every replication step [1,2,3,4,5,6,7,8,9,10,11,12,13,14]. Identifying the host factors and pathways exploited in virus replication and the nature of their contributions and interactions with virus-encoded replication factors represent major challenges and opportunities for understanding and controlling viruses. Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, plant, and animal viruses, has been used as a model system to study gene expression, RNA replication and virus-host interactions of positive-strand RNA viruses [1,2,8, 10,16]. Genomic RNA3 encodes the 3a movement protein, required for infection spread in plants. BMV RNA replication and encapsidation can be recapitulated in the yeast Saccharomyces cerevisiae by expressing the viral replication and/or capsid proteins together with at least one genomic RNA template [20,21,22]. The ability of BMV to duplicate most major replication features of its natural plant hosts in yeast, combined with yeast genetics, has advanced our understanding of BMV replication and virus-host interactions [1,2,9]
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