Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis.

Zhenlu Zhang,Nicholas Catanzaro,Guijuan He,Gil-Soo Han,Arturo Diaz,Jiantao Zhang,George M Carman,Zujian Wu,Lianhui Xie,Xiaofeng Wang

doi:10.1371/journal.ppat.1006988

Abstract

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.

Highlights

Positive-strand RNA viruses [(+)RNA viruses] are the largest of all virus classes and cause numerous important diseases in humans, animals, and plants
In yeast cells with inactive Phosphatidate phosphatase (PAP), total phospholipids, sterol esters, and free fatty acids accumulated to high levels at the expense of TAG
Altered lipid composition substantially promotes cell growth during viral replication. This simultaneously enhances genomic replication of brome mosaic virus (BMV), a well-studied model for examining positive-strand RNA viruses. These results suggest that PAP restricts virus replication by limiting phospholipid synthesis, which is required for viral replication

Summary

Introduction

Positive-strand RNA viruses [(+)RNA viruses] are the largest of all virus classes and cause numerous important diseases in humans, animals, and plants. All of the well-studied (+)RNA viruses have been shown to remodel host intracellular membranes to build viral replication complexes (VRCs) for genomic replication [1,2,3,4]. RNA1- and RNA2-encoded replication proteins 1a and 2a polymerase (2apol) are necessary and sufficient for BMV replication in barley and Nicotiana benthamiana [7,12,13] as well as in yeast [11]. Spherules become VRCs when 2apol and viral genomic RNAs are recruited by 1a during viral replication [11]. Several properties of 1a are required for this process, Host lipin proteins restrict viral replication in yeast and plant including its membrane association domain, an amphipathic α-helix (1a amino acids 392– 407) [22], and its ability to self-interact [23]

Methods

Results

Discussion

Conclusion