Abstract
RNA replication of positive-strand (+)RNA viruses requires the protein-protein interactions among viral replicases and the association of viral replicases with intracellular membranes. Protein A from Wuhan nodavirus (WhNV), which closely associate with mitochondrial membranes, is the sole replicase required for viral RNA replication. Here, we studied the direct effects of mitochondrial membrane lipids (MMLs) on WhNV protein A activity in vitro. Our investigations revealed the self-interaction of WhNV protein A is accomplished via two different patterns (i.e., homotypic and heterotypic self-interactions via different interfaces). MMLs stimulated the protein A self-interaction, and this stimulation exhibited selectivity for specific phospholipids. Moreover, we found that specific phospholipids differently favor the two self-interaction patterns. Furthermore, manipulating specific phospholipid metabolism affected protein A self-interaction and the activity of protein A to replicate RNA in cells. Taken together, our findings reveal the direct effects of membrane lipids on a nodaviral RNA replicase.
Highlights
One universal feature of positive-strand (+)RNA viruses is the assembly of their viral RNA replication complexes, including viral replicase proteins, viral RNA, and host proteins, on host intracellular membranes [1,2,3]
To determine whether Wuhan nodavirus (WhNV) protein A can self-interact in vitro, we used 1 mM of Maltosebinding protein (MBP)-protA to pull down 1 mM of Histagged protein A (His-protA) that was expressed in nucleasetreated rabbit reticulocyte lysates (RRLs) in vitro
MBP-protA was incubated in the chemical cross-linking for 20 min, and the samples were analyzed via SDS-PAGE, revealing one band with molecular weight about 330 kDa (Fig. 2B, lane 4), indicating that protein A can form homodimer
Summary
One universal feature of positive-strand (+)RNA viruses is the assembly of their viral RNA replication complexes (vRCs), including viral replicase proteins, viral RNA, and host proteins, on host intracellular membranes [1,2,3]. During viral RNA replication, these viruses often induce specific intracellular membrane remodeling and lipid biosynthesis modifications via viral replicases [4,5,6]. The viral protein-protein interaction is important for (+)RNA viruses replication [1]. Most (+)RNA viruses encode multiple viral proteins, which work together for the vRCs formation and function [8,9,10,11,12,13]. Some replication proteins from many viruses have activity as individual units, they still require selfinteraction/oligomerization for the complete functionality [14,15,16,17]
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