Hepatitis C virus (HCV) F protein is encoded by the ؉1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein ␣3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the ␣3-binding domain. This ␣3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein.Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperaturesensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit ␣3. The ability of the F protein to bind to ␣3 raises the possibility that the HCV F protein may regulate protein degradation in cells.

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SummaryAkinetes are spore-like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild-type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild-type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real-time quantitative PCR and subsequent testing for akinetespecific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete-expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP-binding cassette (ABC)-type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete-expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.

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