Abstract
Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.
Highlights
Space in the capsids of small viruses is limited and small viruses do not encode every enzymatic activity required for their replication
The subcellular localization of PI4KA and PI4KB is different both enzymes are used by Hepatitis C virus (HCV)
This mechanism was reported for poliovirus (PV), and phosphatidylinositol 4-phosphate (PI4P)-mediated 3Dpol recruitment was proposed as a mechanism for picornaviral and flaviviral replication[39]
Summary
Space in the capsids of small viruses is limited and small viruses do not encode every enzymatic activity required for their replication. Membranous webs are highly enriched in the signaling lipid PI4P (phosphatidylinositol 4-phosphate), yet +RNA viruses do not encode phosphatidylinositol 4-kinases (PI4Ks) Instead, they hijack a human enzyme, either PI4KA or PI4KB ( called PI4K IIIα or PI4K IIIβ)[2,3,4,5,6,7]. Production of PI4P to modify the cholesterol content of membranous webs was demonstrated for the encephalomyocarditis virus (EMCV)[20] Another possible function of PI4P could be direct recruitment of viral effector proteins to the replication sites. GUVs can be prepared from almost any lipid mixture such that their lipid composition resembles that of the specific organelle they are mimicking (here, we use a mixture resembling the Golgi and viral replication organelles) For these reasons GUVs are used to reconstitute and thereby, gain molecular insight into biologically important processes that involve membranes. Additional examples and practical applications can be found in a recent book on model membranes[48]
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