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The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1–191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.

Using immunological and molecular biological techniques, we studied the localization, gene organization, and regulation of expression of the extracellular carbonic anhydrase in the eukaryotic green alga Chlamydomonas reinhardtii. Electron microscopy data using immunogold labelling suggest an association of the protein with the inner face of the cell wall. The same polyclonal antibody was previously used to select an immunoreactive 2.5-kb genomic DNA fragment coding for a portion of the carbonic anhydrase monomer. Using the known carbonic anhydrase cDNA sequence and sequence analysis of the genomic DNA fragment, we have deduced the exon–intron organization of the genomic clone. The similarities between Chlamydomonas and higher plant carbonic anhydrase amino acid sequences and the effect of photoheterotrophic growth on the expression of the algal carbonic anhydrase are also examined. Key words: carbonic anhydrase, Chlamydomonas, immunogold labelling, sequence, expression.