Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

Abstract

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesiclelike structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ϳ4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.All known positive-strand RNA viruses replicate their genomes in association with membranous structures that are formed after the synthesis of viral proteins in infected cells by remodeling membranes from existing intracellular organelles. Different viruses target different organelle membranes (e.g., endoplasmic reticulum [ER], Golgi, endosomes, and mitochondria) and generate different morphological structures on which the replication complexes assemble (37).Among the Picornavirus family members, RNA replication complexes from poliovirus-infected HeLa cells have been the best studied. Heterogeneously sized, vesicle-like structures that cluster in the perinuclear space in infected cells were observed by electron microscopy and described more than 40 years ago. Viral RNA replication occurs on the cytosolic surfaces of the vesicles, which aggregate into clusters (7-9). Little is known about how these structures are formed, although some recent observations have suggested that components of the cellular secretory and/or autophagy pathways are involved (24,39,41).The process of membrane traffic begins at the ER, where polio proteins appear to be synthesized. Rust et al. (36) have visualized, by three-dimensional reconstruction of serial confocal microscope sections, poliovirus protein 2B sequences, and presumably other viral proteins, budding from multiple sites on the ER and showed extensive colocalization of the 2B sequences with COPII coatamer protein.

The phosphatase of regenerating liver 3 (PRL-3) promotes cell migration via Arf-activity dependent stimulation of integrin alpha5 recycling

“…These data indicate that de novo expression of PRL-3, as seen in many forms of cancer, stimulates turnover by functionally hijacking BFA-sensitive Arf1 activation to the turnover machinery of integrins. It is increasingly appreciated that proteins that are expressed in cancer can act in signaling cascades otherwise not influenced by their presence due to subcellular sequestration (Belov et al, 2007). Our data showing that PRL-3 and activated Arf1 are recruited to intracellular membranes strongly support this hypothesis.…”

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

“…There are two types of well-defined families of PI4Ks, type II (PI4KIIa and PI4KIIb) and type III (PI4KIIIa and PI4KIIIb). Previous studies on PV and CVB3 have identified PI4KIIIb as the host enzyme upregulated in viral replication factories (Arita et al, 2011;Belov et al, 2007;Hsu et al, 2010;Lanke et al, 2009). Depletion of PI4KIIIb activity within infected cells by RNA silencing or the use of kinase inhibitors, such as PIK93 (Knight et al, 2006), appeared to block PV and CVB3 viral RNA synthesis and virus replication.…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…The targeting of host factors associated with viral replication complexes (VRCs) such as ADP-ribosylation factor 1 (ARF1), guanine nucleotide exchange factor 1 (GBF1) and phosphatidylinositol kinase 4III (PI4IIIKα/β) has also been shown to inhibit the replication of HCV, several enteroviruses such as picornavirus (PV), Aichi virus (AiV) and Coxsackie virus B3 (CVB3), as well as rhinovirus, mouse hepatitis coronavirus (MHV) and HIV-1 81,[93][94][95][96][97][98][99][100][101] .Some viruses such as DENV and HCV are known to induce up-regulation of lipid synthesis for their replication 54 . Lipid rafts are described to be involved in entry, assembly and/or budding of influenza virus, HCV, VSV, HIV-1, Epstein Barr virus (EBV), Ebola virus (EBOV), Marburg virus (MARV), DENV, West Nile virus (WNV) and Herpes Simplex virus (HSV) ( Table 2).…”