Intellectual Merit. All positive-strand RNA viruses, which include many human, animal and crop pathogens, replicate their RNA genomes on rearranged host intracellular membranes. However, the mechanism whereby these membrane rearrangements are formed is not well defined. The primary objective of the project is to examine the roles of host acyl-CoA binding protein (ACBP) in the assembly and function of viral replication complexes, or viral spherules, of Brome mosaic virus (BMV) using a combination of genetic, biochemical and cell biology approaches. ACBP is highly conserved in all eukaryotes, specifically binds to long chain fatty acyl-CoA, and plays major roles in cellular lipid synthesis. Lack of ACBP severely inhibits BMV replication and results in formation of aberrant viral spherules with a significantly reduced diameter paralleled by a 5-fold increase in frequency compared to those in wild type cells. The specific objectives are: (1). Determining the role of 1a-ACBP interaction in BMV spherule formation and replication. (2). Determining the roles of ACBP in modulating lipid synthesis during BMV replication. The results from this project will better elucidate relations between lipid synthesis and viral replication as well as the microenvironment of the viral replication complexes. Such understanding would advance fundamental knowledge of viral replication mechanisms and virus-host interactions common to positive-strand RNA viruses, and allow identification of potential targets for virus control in humans, animals and plants. Broader impact: ACBP is essential in mammals and this project should stimulate broader interests in testing its roles in replication of human and animal viruses. Besides training a post-doctoral research associate, the PI will continue to involve and train high school and undergraduate students from the Rio Grande Valley (Texas), where more than 85% population are Hispanic.
Many important human, animal and plant pathogens are positive-strand RNA viruses, causing hepatitis, hemorrhagic fever, and common cold, among other widespread and costly diseases as well as billion-dollar agricultural losses per year. Genomic replication of these viruses relies on the viral ability to remodel host membranes for form host membrane-invaginated viral replication complexes (VRCs). However, mechanisms of such membrane rearrangements, host genes involved in such process, and microenvironment of such VRC(s) are not well defined. In this project, we are dissecting the functional roles of host acyl-CoA binding protein (ACBP) in brome mosaic virus (BMV)-mediated VRCs. ACBP is highly conserved among eukaryotes from yeast to plant to human. What we learned should be applicable to other viruses. ACBP is involved in lipid homeostasis, particularly fatty acids. In cells lacking ACBP, BMV replication was inhibited 30-fold and BMV VRCs are 50% in size but 4-fold more in numbers compared to those in wild-type cells. Supplementing unsaturated fatty acids can largely complement all replication defects, indicating the inhibited BMV replication is through ACBP’s function in lipid metabolism. BMV replication modulates phospholipid synthesis and composition by promoting phophatidylcholine (PC), a feature has been reported during other viral infections. We found that ACBP is required for BMV-promoted PC synthesis. In addition, we found BMV interacted with a key enzyme involved in PC synthesis, highlighting that BMV may recruit the enzyme to viral replication sites to synthesize a pool of PC designated for viral synthesis. Among others, we also found that altered phospholipid composition also affected VRC sizes. Through the project, a graduate student and a post-doc have been trained. Four undergraduate students participated in the project. These included 3 minority students and two of them are females. In addition, a week-long training camp was held in the summer 2013 and 2014 for minority students, consistent with PI’s long-term objective to provide training and attract minority students into the field of life sciences thus diversify the work force in the field.
