Date Published: August 27, 2018
Publisher: Public Library of Science
Author(s): Ekaterina G. Viktorova, Jules A. Nchoutmboube, Lauren A. Ford-Siltz, Ethan Iverson, George A. Belov, Ralf Bartenschlager.
Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system.
The positive strand RNA ((+)RNA) viruses of eukaryotes universally assemble their RNA replication machinery in association with specialized membranous domains, featuring unique lipid and protein composition [1–3]. It is hypothesized that membranes may facilitate replication by increasing local concentration of the viral proteins, providing a scaffold for assembly of the multi-subunit replication complexes, and/or by hiding the dsRNA replication intermediates from cellular sensors of infection . To trick the infected cells into building new membranous structures, viruses have to reorganize the complex network of cellular pathways controlling lipid synthesis, catabolism and membrane trafficking. Yet, in spite of the central role of the membranous replication organelles in the life cycle of (+)RNA viruses, our knowledge about the mechanistic details of their formation in most viral systems is very limited, and the experimental evidence supporting their importance for specific replication steps is scarce.
The rapid development of the membranous replication structures is one of the longest known but still enigmatic cellular manifestations of picornavirus infection. In cells infected with poliovirus or a related Coxsackie B3 virus, the virus-induced membranes appear as early as 2 h p.i. at the ER-Golgi interface and continue to grow throughout the replication cycle, transitioning from sponge-like membranous clusters to assemblages of double membrane vesicles [11, 74–76]. The unique morphology of the replication organelles implies that mechanism(s) of their formation and/or their composition are different from those supporting membrane architecture in non-infected cells. These novel membranous structures, which may occupy most of the cytoplasmic space by the end of infection, are known to harbor actively replicating viral RNA, and thus are generally referred to as replication organelles. Later in infection, progeny virions are found both inside and outside the membranous vesicles, and the data suggest that the double membrane vesicles accumulated by the end of infection may facilitate virion maturation and spread [22, 65, 77, 78]. Still, our understanding of the mechanistic contribution of the membranous matrix in the viral life cycle is mostly speculative. Development of membranous replication complexes is the hallmark of infection of all (+)RNA viruses of eukaryotes, suggesting that membrane association of the RNA replication and/or virion assembly machinery provides specific benefits in the cytoplasmic environment. On the other hand, it cannot be excluded that massive production of membranes is related to the cellular antiviral response aimed at blocking accessibility of the cellular translational apparatus or other metabolic resources necessary for virus propagation.