Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites Jiantao Zhanga,1, Zhenlu Zhanga,b, Vineela Chukkapallic, Jules A. Nchoutmboubed, Jianhui Lia, Glenn Randallc, George A. Belovd, and Xiaofeng Wanga,2 aDepartment of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; bDepartment of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China; cDepartment of Microbiology, The University of Chicago, Chicago, IL 60637; and dVirginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved January 12, 2016 (received for review October 6, 2015) All positive-strand RNA viruses reorganize host intracellular mem- expression of 1a alone in yeast induces spherule formation (6), branes to assemble their viral replication complexes (VRCs); how- which requires 1a’s amphipathic α-helix (1a amino acids 392–407) ever, how these viruses modulate host lipid metabolism to (8), helix A, and 1a–1a interactions (9, 10). In addition, several accommodate such membrane proliferation and rearrangements is host proteins, including membrane-shaping reticulons (RTNs) not well defined. We show that a significantly increased phospha- (11) and an ESCRT (endosomal sorting complex required for tidylcholine (PC) content is associated with brome mosaic virus transport) component, Snf7p (sucrose nonfermenting7) (12), are (BMV) replication in both natural host barley and alternate host recruited by 1a to form spherules. yeast based on a lipidomic analysis. Enhanced PC levels are primarily Similar to other (+)RNA viruses, BMV promotes host lipid associated with the perinuclear ER membrane, where BMV replica- synthesis and requires balanced lipids for the formation and ac- tion takes place. More specifically, BMV replication protein 1a interacts tivity of VRCs. Expression of 1a in yeast induces a ∼30% increase with and recruits Cho2p (choline requiring 2), a host enzyme in- in total fatty acids (FAs) per cell (13). BMV also requires a high volved in PC synthesis, to the site of viral replication. These results level of unsaturated FA (UFA) because a ∼12% decrease of suggest that PC synthesized at the site of VRC assembly, not the UFAs in the yeast ole1w mutant blocks its replication more than transport of existing PC, is responsible for the enhanced accumula- 20-fold (13). In addition, deleting host ACB1 (Acyl-CoA-binding 1) tion. Blocking PC synthesis by deleting the CHO2 gene resulted in gene results in formation of spherules that are smaller in size VRCs with wider diameters than those in wild-type cells; however, but are in greater number than in wild-type (WT) cells (14). BMV replication was significantly inhibited, highlighting the critical ACB1 encodes acyl-CoA binding protein, which binds long-chain role of PC in VRC formation and viral replication. We further show fatty acyl-CoAs and is involved in maintaining lipid homeostasis. that enhanced PC levels also accumulate at the replication sites of Supplemented long-chain UFAs largely complement the BMV hepatitis C virus and poliovirus, revealing a conserved feature among replication defects in cells lacking ACB1, indicating that the a group of positive-strand RNA viruses. Our work also highlights a altered lipid composition is primarily responsible for BMV repli- potential broad-spectrum antiviral strategy that would disrupt PC cation defects (14). synthesis at the sites of viral replication but would not alter Cellular membranes are mainly composed of phospholipids, and cellular processes. in particular, phosphatidylcholine (PC) constitutes ∼50% of total phospholipids (15). PC is synthesized via the CDP–DAG (cytidine positive-strand RNA viruses | viral replication complexes | diphosphate–diacylglycerol) and Kennedy pathways in eukaryotes virus–host interactions | virus control | phospholipids (16). PC synthesis is significantly enhanced during infection of ll positive-strand RNA viruses [(+)RNA viruses], which in- Significance Aclude numerous important human, animal, and plant path- ogens, share similar strategies for genomic replication. A highly Positive-strand RNA viruses [(+)RNA viruses] include many im- conserved and indispensable feature of their replication is the portant human, animal, and plant pathogens. A highly con- proliferation and reorganization of host cellular membranes to as- served and indispensable feature of (+)RNA virus infection is semble viral replication complexes (VRCs). Despite this cen- that these viruses proliferate and reorganize host membranes tral importance, it is largely unknown how cellular membranes to assemble viral replication complexes (VRCs). We show that are rearranged by the viral replication proteins and how cellular brome mosaic virus (BMV) stimulates phosphatidylcholine (PC) lipid metabolism is modulated to accommodate membrane pro- synthesis at the viral replication sites. BMV recruits a host en- liferation and remodeling. zyme involved in PC synthesis to support proper VRC formation Brome mosaic virus (BMV) serves as a model for understanding and genomic replication. We further show that hepatitis C virus VRC formation of (+)RNA viruses (1). BMV is the type member and poliovirus also promote accumulation of PC at the viral of the plant virus family Bromoviridae and a representative member replication sites, revealing a feature common to a group of of the alphavirus-like superfamily, which includes many human, (+)RNA viruses. This virus-specific step can be targeted to de- animal, and plant-infecting viruses (2). BMV encodes two repli- velop a broad-spectrum antiviral strategy with the least side cation proteins, 1a and 2apol.2apol serves as the replicase, whereas effects on host growth. 1a has an N-terminal methyltransferase domain (3, 4) and a C-terminal ATPase/helicase-like domain (5). Together, 1a and Author contributions: J.Z., G.R., G.A.B., and X.W. designed research; J.Z., Z.Z., V.C., J.A.N., and pol J.L. performed research; X.W. coordinated the research; J.Z., Z.Z., V.C., J.A.N., J.L., G.R., G.A.B., 2a are necessary and sufficient for BMV replication. BMV and X.W. analyzed data; and J.Z., G.R., G.A.B., and X.W. wrote the paper. Sac- induces vesicular structures in its surrogate host, the yeast The authors declare no conflict of interest. charomyces cerevisiae ,anditsnaturalhost,barley(6,7).These This article is a PNAS Direct Submission. structures, termed spherules, have been shown to be the VRCs in 1 pol Present address: Department of Pharmacology and Toxicology, The University of Ari- yeast as 1a, 2a , and nascent viral RNAs reside in the interior zona, Tucson, AZ 85721. of these compartments. Spherules are invaginations of the outer 2To whom correspondence should be addressed. Email: [email protected]. perinuclear endoplasmic reticulum (ER) membrane into the ER This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lumen and are about 60–80 nm in diameter (6). Remarkably, 1073/pnas.1519730113/-/DCSupplemental. E1064–E1073 | PNAS | Published online February 8, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Downloaded by guest on September 28, 2021 Dengue virus (DENV) (17), Flock House virus (FHV) (18), and the 1a signal (∼60% of the population), PC colocalized with 1a in PNAS PLUS poliovirus (19, 20). A 70% and 35% increase of total PC levels >92% of cells. was recorded in DENV-infected mosquito cells (17) and FHV- PE is the substrate of PC in the CDP–DAG pathway, and in infected Drosophila cells (18), respectively. At the peak time of addition, PE is enriched at the viral replication sites of several poliovirus replication in HeLa cells, a ∼37% increase of PC (+)RNA viruses (24). To determine whether PE distribution is content was observed after a 30-min chase (19). It was further affected by BMV, we used a PE biosensor, duramycin peptide found that poliovirus promotes the import of FAs, which were (25). In the absence of BMV, PE is predominantly present in the subsequently channeled to the viral replication sites. In addition, cytoplasm. We found that a portion of PE signal, albeit very FAs were mainly incorporated into PCs (20). weak, colocalized with that of 1a in the presence of BMV rep- One critical question based on the aforementioned research is lication (Fig. S2). whether the enhanced PC is synthesized in association with the VRCs or elsewhere in cells and subsequently transported into BMV Promotes PC Accumulation at the Site of Viral Replication in the VRCs. If PC is produced in association with the VRCs, what Plants. To verify that BMV promotes an increase in PC levels key enzymes are recruited? We report here that several (+)RNA during natural infection, we analyzed phospholipid compositions viruses, including BMV, hepatitis C virus (HCV), and poliovirus, in mock- and BMV-inoculated barley systemic leaves at 7 d post- promote significantly enhanced accumulation of PC content at inoculation (dpi), a time when the systemic leaves started to show the viral replication sites, revealing a common feature of viral symptoms. Similar to BMV-replicating yeast cells, the amount replication among a group of (+)RNA viruses. We further dem- of total phospholipid and PC increased by ∼24% and ∼29%, re- onstrate that BMV 1a interacts with and redistributes the host spectively, in BMV-infected plants relative to mock-inoculated enzyme, Cho2p (choline requiring 2), to the viral replication sites. plants (Fig. 1E). In cell wall-free protoplasts prepared from mock- As Cho2p converts phosphatidylethanolamine (PE) to PC in the inoculated leaves, the PC signal was evenly distributed throughout CDP–DAG pathway, the relocalization of Cho2p suggests the the cytoplasm but was excluded from the nucleus (Fig. 1F). A more VRC-localized PC synthesis. Deleting CHO2 inhibits BMV rep- intense PC signal was occasionally detected surrounding the nu- lication up to 30-fold and results in formation of spherules that are cleus (Fig. 1F, Top Left). Agreeing with previous reports showing larger than those of WT cells.