Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Abstract

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K⁺) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K⁺ channel proteins consisting of 82 amino acids. The Kcv_ATCV-1 protein has similarities to the family of two transmembrane domain K⁺ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K⁺ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from Kcv_ATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K⁺ channels.

Keywords: Chloroviruses; Kcv channels; algal viruses; potassium ion channels.

Figure A1

DNA sequence alignment of unique SAG chlorovirus kcv genes. DNA sequences were aligned using the Geneious Alignment algorithm from Geneious 11.0.5 software. Green color indicates identical nucletides among all sequenced strains. Different shades from olive to orange denote different degrees of conservation with olive color being the most conserved.

Figure A1

DNA sequence alignment of unique SAG chlorovirus kcv genes. DNA sequences were aligned using the Geneious Alignment algorithm from Geneious 11.0.5 software. Green color indicates identical nucletides among all sequenced strains. Different shades from olive to orange denote different degrees of conservation with olive color being the most conserved.

Figure A1

DNA sequence alignment of unique SAG chlorovirus kcv genes. DNA sequences were aligned using the Geneious Alignment algorithm from Geneious 11.0.5 software. Green color indicates identical nucletides among all sequenced strains. Different shades from olive to orange denote different degrees of conservation with olive color being the most conserved.

Figure 1

Kcv_ATCV-1 sequence alignment with the sequence of the prototype chlorovirus K⁺ channel Kcv_PBCV-1 by ClustalW. The slide helix, outer transmembrane (TMD1) turret, pore helix, selectivity filter, and inner transmembrane (TMD2) of the Kcv_PBCV-1 are highlighted by the horizontal lines. Asterisks indicate positions which have a fully conserved residue. A colon indicates conservation between amino acids with strongly similar properties. A period indicates conservation between amino acids with less similar properties.

Figure 2

Amino acid alignment of SAG chlorovirus unique Kcv proteins using Geneious 11.0.5 software. The number in brackets at the end of each protein is the number of times that the protein had an identical sequence of the 103 viruses examined. Score matrix is Identity. Green color denotes identical amino acids. Other shades of amino acids indicate a level of conservation from olive color as being the most conserved to white color not conserved.

Figure 3

Phylogenetic tree of Kcv proteins from 26 SAG chloroviruses. PhyML, which is Maximum likelihood, with the default settings was used to construct the tree. The branch length shows dissimilarity between strains and the values on the branches are the number of changes. The viruses in blue represent the new Kcv proteins reported in this manuscript. Viruses MT325 representing a Pbi virus and PBCV-1 representing an NC64A virus are in green. Additional information on each of the Kcv proteins from the SAG viruses, including where they were isolated and the number of times that protein sequence appeared, is included in Table A1.

Figure 4

Two of the newly discovered K⁺ channels are active. Characteristic single-channel fluctuations (a), mean single-channel I/V relations (b), and mean open probabilities (c) of Kcv_NTS (top row), Kcv_GNLD22 (middle row), and Kcv_Can0610SP (bottom row) at ±120 mV. The closed (c) and open (o) levels are indicated along the current traces. Data are means ±s.d. from ≥ 3 independent recordings of channels in the same row. Data were recorded in a DPhPC bilayer with symmetrical 100 mM KCl, 10 mM HEPES, pH 7 in cis and trans chamber.