Chitin synthesis by Chlorella cells infected by chloroviruses: Enhancement by adopting a slow-growing virus and treatment with aphidicolin.

Chlorella viruses or chloroviruses contain a gene that encodes an enzyme that catalyzes chitin synthesis. This gene is expressed early in viral infections to produce chitin on the outside of the Chlorella cell wall. Interestingly, chitin synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO2 into useful materials. However, during the final viral infection stage, the host cells are completely lysed, so chitin should be harvested before cells lyse. To increase chitin yields, slow-growing chlorovirus isolates were adopted and the viral replication process was modified with an inhibitor of DNA synthesis. The accumulation of chitin on the surface of Chlorella cells infected with one of nine chlorovirus isolates carrying the chitin synthase gene was compared with that of CVK2 (a standard virus)-infected cells. Chlorella cells infected with CVNF-1 (a slow-growing virus) accumulated chitin over the entire cell surface within 15 min post-infection (p.i.), and chitin continued to accumulate for up to 8 h p.i. before cells lysed. This was 2-fold longer than the chitin-accumulation period for cells infected with CVK2. The addition of aphidicolin delayed the progression of the virus replication cycle and extended the chitin-accumulation period of CVNF-1-infected cells to 12 h p.i. before cells lysed. Additionally, chitin production in the aphidicolin-treated CVNF-1-infected cells was approximately 6-fold higher than in CVK2-infected cells not treated with aphidicolin. Thus, chitin synthesis in a Chlorella-virus system may be prolonged by using slow-growing viral isolates treated with aphidicolin.

Prolonged synthesis of hyaluronan by Chlorella cells infected with chloroviruses.

Hyaluronan (HA) synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO2 into useful materials. However, at the final stage of viral infection, infected host cells are completely lysed, and thus HA should be harvested before cell lysis. In the current study, two methods were investigated to improve the yield of HA: (i) adopting slow-growing chlorovirus isolates and (ii) modification of the virus replication process using an inhibitor of DNA synthesis, aphidicolin. Compared with Paramecium bursaria Chlorella virus type 1 (PBCV-1), the prototype chlorovirus, slow-growing virus isolates (CVO1 and CVTS1) produced a 1.5 times higher concentration of HA in infected Chlorella cultures. Furthermore, addition of aphidicolin, an inhibitor of DNA synthesis, delayed virus replication and increased the final concentration of HA 1.5-fold that of cultures without the addition of aphidicolin. Therefore, a 2- to 3-fold increase in the yield of HA by the Chlorella-virus system was attained by using slow-growing viral isolates and the addition of aphidicolin.

A potassium channel protein encoded by chlorella virus PBCV-1.

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

[The resistance of the DNA of cyanophage LPP-3 to the action of different restriction endonucleases]. / Rezistentnost' DNK tsianofaga LPP-3 k deistviiu razlichnykh éndonukleaz restriktsii.

Data on the study of structure peculiarities of cyanophage LPP-3 DNA are presented in the work. The length of cyanophage DNA calculated by means of the enzymatic hydrolysis by restrictases is 40 +/- 3.5 thou. pairs of bases. Cyanophage LPP-3 DNA was hydrolysed by more than 50 different restrictases. As a result of screening it was found out that the great number of restrictases, which recognized hexanucleotide sequences did not hydrolyze DNA of cyanophage LPP-3. A considerable deviation of the number of the observed sites of restriction from their theoretically expected number for restrictases Hae III and Cfr 131 was established. Restrictases-isoschisomeres with different sensitivity to the methylation of the recognition sites--Msp I, Hpa II and Sau 3A, MboI and DpnI were used to check the availability of methylated bases in LPP-3 DNA. Absence of methylated adenine in the site GATC and methylated cytosine in the second position of the site CCGG were established. The results obtained permit supposing that the expressed counterselection by the sites of recognition of many restriction endonucleases takes place in cyanophage LPP-3 DNA. It is supposed that apparently, this method of protection of its genome in LPP-3 is one of most important but the inconsiderable percentage of site-specific methylation of the virus DNA cannot be completely excluded.