Two distantly related algal viruses of the family Phycodnaviridae encode K+ channel-like genes. Paramecium bursaria Chlorella virus encodes Kcv, the first known viral K+ channel protein. Kev, a second putative viral K+ channel was derived from the genome of Ectocarpus siliculosus virus (EsV-1). The sequence of EsV-1 open reading frame 223 encodes 124 amino acids with high homology to the Chlorella virus channel Kcv. Expression of the pore region of Kev in a chimeric protein demonstrated that the Kev pore is indeed functional. Even though, Kev wildtype protein failed to induce a K+ selective conductance in heterologous expression systems HEK293 cells, CHO cells, and Xenopus laevis oocytes. The failure to detect K+ channel activity at the plasma membrane of Kev expressing cells is most probably due to the intra-cellular distribution of Kev in these cells: confocal microscopy of HEK293 cells expressing Kev-GFP fusion protein shows fluorescence signals with punctuated maxima. In contrast, Kcv-GFP fluorescence accumulates in tubular membranous structures. Kev mutants with their carboxy-terminal helix extended by 3 or 6 amino acids show a Kcv-like pattern of distribution demonstrating that the length of the carboxy-terminal helix is crucial for the cellular distribution of the channel protein. These findings indicate, that Kev function in nature might occur either at the thin membrane of the host cell endoplasmic reticulum (ER) or at the ER-derived internal membrane of Ectocarpus virus particles. The data presented in this thesis suggest that Chlorella virus channel Kcv is localised in the virion: (i) Kcv mRNA is synthesised during the late phase of viral replication when assembly of new virus particles takes place. (ii) The membrane potential of the host cells depolarises during infection of Chlorella NC64A cells by Chlorella viruses, as monitored using the potential-sensitive probe Bis-Oxonol. This host cell depolarisation exhibits a pharmacology parallel to both, viral replication, and Kcv activity in heterologous expression systems. Thus, Kcv activity seems to be essential during the first minutes of Chlorella virus infection. | English |