Research Article: Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

Date Published: May 21, 2018

Publisher: Public Library of Science

Author(s): Sravani Banerjee, David Aponte-Diaz, Calvin Yeager, Suresh D. Sharma, Gang Ning, Hyung S. Oh, Qingxia Han, Masato Umeda, Yuji Hara, Robert Y. L. Wang, Craig E. Cameron, Glenn Randall.

http://doi.org/10.1371/journal.ppat.1007086

Abstract

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

Partial Text

Myriad cellular mechanisms exist to thwart viral infection [1–4]. These mechanisms are triggered when a cellular pattern recognition receptor (PRR) engages a virus-associated molecular pattern, for example 5’-triphosphorylated RNA, the absence of 2’-O-methylation of the mRNA cap, double-stranded RNA, among many others [1–4]. PRRs are located at every portal of viral entry into a cell but are particularly abundant in the cytoplasm, the site of replication of most RNA viruses, especially positive-strand RNA viruses. RNA viruses have evolved multiple mechanisms to escape host innate immunity [1–4]. Some mechanisms are specific, for example the use of virus-encoded protein(s) to bind and/or to degrade a PRR [1–4]. One generic approach exploited by positive-strand RNA viruses may be the use of a replication organelle for genome replication, which limits surveillance by cellular antiviral defenses [5], although the need to evade host defenses in cell culture may not be absolute [6].

Phosphoinositides distinguish one organelle from another, couple activation of protein/enzyme function to appropriate cellular localization, and, along with small GTPases and their effectors, enable directional trafficking of proteins and membranes in the cell [17]. In the context of this long-established paradigm of cell biology, the discovery that picornaviruses use PI4P to target proteins to sites of genome replication was logical but also stunning [16]. Since this transformative discovery was made, many laboratories have strived to fill in the gaps between virus entry and formation of the PI4P-rich replication organelle. In the cell, PI4P biogenesis begins with GEF recruitment to the membrane to produce Arf-GTP, which, in turn, recruits effectors that ultimately lead to the recruitment and/or activation of a PI4 kinase [19]. In the specific case studied here, how GBF1 is recruited to membranes and the number and identity of the factors between Arf1-GTP and PI4KB is unclear. However, our studies invoke a requirement for 3CD instead of other P3-derived proteins (Fig 11A and 11B).

 

Source:

http://doi.org/10.1371/journal.ppat.1007086