Hepatoma polarization limits CD81 and hepatitis C virus dynamics.

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.

Primary hepatocytes as targets for hepatitis C virus replication.

Much of our current understanding of hepatitis C virus (HCV) replication has hailed from the use of a small number of cloned viral genomes and transformed hepatoma cell lines. Recent evidence suggests that lipoproteins play a key role in the HCV life cycle and virus particles derived from the sera of infected patients exist in association with host lipoproteins. This report will review the literature on HCV replication in primary hepatocytes and transformed cell lines, focusing largely on host factors defining particle entry.

Interaction of PLD1b with actin in antigen-stimulated mast cells.

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.

The over-expression of the wild type or mutant forms of the presenilin-1 protein alters glycoprotein processing in a human neuroblastoma cell line.

Mutations in the presenilin proteins (PS1 and PS2) are responsible for more than 70% of the cases of the familial form of Alzheimer's disease (FAD). The proteins are expressed in the cell at a low level, primarily in the endoplasmic reticulum and cis Golgi, where they have been proposed to play a role in protein processing. As protein glycosylation is a key post-translational event that occurs within the Golgi, we have investigated the effect of altered PS1 expression levels on the protein glycosylation pattern using the SH-SY5Y human neuroblastoma cell line. In cells over-expressing either the wild type or mutant (M146L) PS1-FAD proteins, there was a decrease in the expression levels of protein-bound alpha2,3-linked sialic acid residues at the level of the cell membrane. This was particularly manifest as a significant decrease in the expression of the polysialic acid chain that is linked to the core oligosaccharide of the neural cell adhesion molecule in an alpha2,3 bond. These results suggest that the over-expression of either the wild type or mutant PS1 disturbs glycoprotein processing within the Golgi.