Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

Abstract

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.Hepatitis C virus (HCV) is an enveloped positive-stranded RNA virus and the sole member of the genus Hepacivirus within the Flaviviridae. Approximately 170 million individuals are infected with HCV worldwide, and the majority are at risk for developing serious progressive liver disease. The HCV RNA genome of approximately 9.6 kb encodes a polyprotein of around 3,000 amino acids, which is cleaved by viral and cellular proteases to generate the structural and nonstructural (NS) proteins. The amino terminus of the polyprotein sequence contains the structural proteins including the core, the envelope glycoproteins (GPs) E1 and E2, and p7. The NS proteins including NS2 through NS5 are located at the carboxy terminus of the polyprotein. Much of our current understanding of HCV replication has been gained through the use of genomic replicons (reviewed in reference 7). The recent development of an infectious system allowing the generation of HCV particles in cell culture (HCVcc) has enabled the complete viral life cycle to be explored (63,99,110).

Entry and replication of recombinant hepatitis C viruses in cell culture

“…How the virus triggers a cellular response leading to its internalization and delivery to a compartment favourable for fusion remains incompletely understood and requires systematic studies of the signalling cascades regulated after virus attachment onto the cell surface. Several groups attempted to tackle this issue, by screening libraries of kinases for those that are important for HCV entry [141] or by targeting specific signalling molecules using genetic or pharmaceutical approaches [42,173]. cells that express all HCV receptors (e.g.…”

Host–virus interactions during hepatitis C virus infection: a complex and dynamic molecular biosystem

“…Recently it has been established that the E1 and E2 viral proteins interact with four known host receptors CD81, claudin-1, SRB-1 and occludin and their interaction can also be mediated by GAGs, the LDL receptor, and both DC and liver cell specific-SIGN. [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] The next step involves the fusion of the virion particle with membranes of the host cell and subsequent uncoating and release of the components of the virion particle that include the positive strand HCV genomic RNA, some HCV non-structural proteins, including NS5A, and, in some cases, host proteins, including low density lipoproteins (LDLs). 25,28,29 The HCV RNA then moves to ribosomes where the HCV polypeptide is translated and processed as previously mentioned.…”

Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

“…These include the tetraspanin CD81 (30), scavenger receptor class B type I (SR-BI) (32), and more recently described, the tight junction components claudin 1 (CLDN1) (13) and occludin (OCLN) (27,31). It is thought that the viral envelope glycoproteins E1 and E2 interact with these four (co)receptors sequentially and that these interactions are orchestrated by cell signaling processes (6,14,28). SR-BI may act early and the tight junction components may act later in this process (11,13,21,24,42).…”