Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication

Abstract

A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

“…However, cellular studies using RNAi-mediated knockdown indicated that PI4KA is responsible for the maintenance of the PM PtdIns(4)P and PtdIns(4,5)P 2 pools during strong PLC activation (10) and predicted that complete blockade of this enzyme may interfere with a number of signaling processes that require the presence of these lipids in the PM. The conclusion drawn from these studies was that cells tolerate a significant level of knockdown of PI4KA because even a small amount of enzyme is able to maintain its housekeeping functions, whereas HCV replication is strongly inhibited even by a 50% reduction of the level of this enzyme (15,17,39,40). This has lent strong support to the notion that there is a therapeutic window for inhibiting HCV replication without affecting cellular functions.…”

“…Synthesis of selective PI4KA inhibitors has been motivated by the prospect that such inhibitors might be useful to fight HCV infection. RNAi studies identified PI4KA as a mandatory host factor in HCV replication, and those studies suggested that PI4KA down-regulation is reasonably tolerated by cultured cells (15,17,39,40). In addition, it has been reported that HCV infection can induce the expression of PI4KA and increase PtdIns(4)P levels in humans (39).…”

“…An unexpected and much needed stimulus in PI4K research was brought about by the finding that PI4KA is an essential host factor for hepatitis C virus (HCV) replication, reported in several simultaneous studies using RNAi screens (15)(16)(17)(18)(19). In parallel studies, it was also shown that several small RNA enteroviruses reprogrammed the endoplasmic reticulum-Golgi trafficking machinery to serve their replication needs and used another PI4K, PI4KB, in the process (14,20).…”

Hepatitis c virus and host cell lipids: An intimate connection

“…132 The importance of PI4K-IIIα for HCV replication is well established, because this kinase has been identified in several siRNA-based screens as one of the top hits, regardless of the viral genotype and the experimental system. [133][134][135][136][137][138] In contrast, the role of the Golgi-resident PIK4-IIIβ is still controversially debated; it appears to contribute to replication of genotype 1 replicons, but not to replication of the JFH-1 genotype 2a isolate. 131,135,136 Nevertheless, the two isoforms of the kinase are believed to perform complementary, non-redundant tasks, because silencing of each gene decreases the levels of PI(4)P in HCV replicon cells, 81 and because the inhibitory effect mediated by PI4K-IIIα knockdown on HCV replication cannot be rescued by overexpression of PIK4-IIIβ.…”

A new paradigm in viral resistance

“…Specific cellular factors used by several picornaviruses (such as PV and CVB3) and flaviviruses (such as HCV) have been identified and their mechanistic roles and potential as therapeutic targets are currently being evaluated. Of note is that a microenvironment rich in phosphatidylinositol-4-phosphate (PI4P), established through the recruitment of the phosphatidylinositol-4-kinase IIIα (PI4KIIIα) and/or IIIβ (PI4KIIIβ), is crucial for replication of several of these viruses [7][8][9][10][11][12][13]. Therefore, inhibitors targeting these components would offer broad-acting therapeutic potential.…”