Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.

Abstract

Flockhouse virus (FHV) is a member of the nodavirus group ofpositive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNAdependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon in of glycerophosphoilpid (GPL) Complete replication of FHV RNA by the membraneassociated FHV replicase depended on the absence of membrane-disrupting detergents and the presentation of template RNA in combination with Lipofectin (BRL), a reagent commonly used to introduce RNA and DNA into membrane compartments (4, 7). In the presence of detergent or absence of Lipofectin, added FHV RNA was copied to give (-)-strand RNA, but no new (+)-strands were synthesized.Lipofectin and its analogs are mixtures of a specific glycerophospholipid (GPL) and a cationic lipid. We have now analyzed the contribution of each component and found that only the GPL is required for complete FHV RNA replication in vitro. We further show that a number of GPLs support complete FHV RNA replication and that the activity of effective GPLs is dependent on the presence and structure of both the polar head group and the acyl chains. The amount of GPL required to support (+)-strand synthesis increases as more FHV replicase extract is added to the reaction. This result, the dispensability of the cationic lipid, and other observations suggest that the role of GPL in facilitating (+)-strand RNA synthesis is independent of delivery of the initial template RNA. Since the RNA replication complexes of most or all (+)-strand RNA viruses are membraneassociated and (+)-strand RNA synthesis has generally been the block to complete RNA replication in vitro, the effects of GPL on FHV RNA replication might have general implications for many viruses. MATERIALS AND METHODSPhospbolrps and Related Compounds. The following compounds were obtained from Sigma in the highest purity grades available: dimethyldioctadecylammonium bromide (DDAB); L-a-phosphatidic acid (PA, from egg yolk); L-a-phosphatidylcholine (PC, from egg yolk); L-a-phosphatidylethanolamine, dioleoyl (PE, synthetic); L-a-phosphatidyl-racglycerol, dioleoyl (PG, synthetic); L-a-phosphatidylinositol (PI, from soybean); L-a-phosphatidyl-L-serine (PS, from bovine brain); sphingomyelin (SPM, from bovine brain); cholesterol; 1,2-dimyristoyl-rac-glycerol [14:0 (i.e., carbons: double bonds in each acyl chain = 14:0)]; 1,2-dioleoyl-racglycerol [18:1 (double bond position = cis-9)

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