Dwight E. Burbank

Growth cycle of a virus, PBCV-1, that infects Chlorella-like algae.

The growth and purification of milligram quantities of a large double-stranded DNA virus, PBCV-1, which replicates in a Chlorella-like alga is described. The virus had an adsorption rate constant of ca. 5 x 10(-9) ml/min, a latent period of 150 to 180 min, and a burst size of 200 to 350 PFUs when the host was actively growing in the light. The eclipse period was 30 to 50 min shorter than the latent period. PBCV-1 also replicated in dark grown Chlorella but the burst size was reduced ca. 50%. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, had no effect on viral replication. Thus viral replication does not require host photosynthesis. Viral infection rapidly inhibited both growth and CO2 fixation of the host Chlorella.

Virus Infection of Culturable Chlorella-Like Algae and Dlevelopment of a Plaque Assay

Four distinct viruses with double-stranded DNA are known to replicate in Chlorella-like algae symbiotic with hydras and paramecia. An attempt was made to infect a number of cultured Chlorella strains derived from invertebrate hosts with these viruses. One of the viruses, PBCV-1, replicated in two of the algal strains. Restriction endonuclease analysis of the viral DNA showed that the infectious progeny virus was identical to the input virus; thus, Kochs postulates were fulfilled. Viral infection of the two Chlorella strains has allowed the large-scale production of a eukaryotic algal virus and the development of a plaque assay for the virus.

Infection of a chlorella-like alga with the virus, PBCV-1: Ultrastructural studies☆

Ultrastructural studies revealed that the virus, PBCV-1, adsorbs to the surface of the Chlorella-like green alga NC64A and enzymatically digests a portion of the host cell wall. The viral DNA is then released into the interior of the cell leaving an empty capsid on the surface. Thus uncoating of the viral genome occurs at the surface of its host. PBCV-1 also adsorbs to and digests the host wall of either heat-killed, methanol-extracted, or purified cell wall fragments.

DNA synthesis in a Chlorella-like alga following infection with the virus PBCV-1☆

Infection of the unicellular, eukaryotic Chlorella-like alga NC64A by the large dsDNA virus, PBCV-1, resulted in a threefold increase in total DNA by 4 hr post infection. Viral infection rapidly inhibited host DNA synthesis which was followed by the degradation of the host chloroplast and nuclear DNA. Viral DNA synthesis began 30 to 40 min after infection and was dependent on de novo protein synthesis. Thus, the virus does not carry all of the components required to form a functional viral DNA polymerase into the cell.

Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra viridis.

The isolation and characterization of a virus (designated HVCV) from the symbiotic Chlorella-like green alga present in Hydra viridis (Florida strain) are described. The large polyhedral viral particle sedimented at ca. 2600 S in sucrose density gradients and had a buoyant density in CsCl of 1.295 g/ml. The virus contained at least 19 polypeptides (MW range, 10,300 to 82,000) and the major polypeptide (MW, 46,000) was a glycoprotein. The viral genome consisted of a double-stranded DNA with a buoyant density in CsCl of 1.7114 g/ml (52% GC) and a molecular weight of ca. 136 x 10(6).

Structural proteins and lipids in a virus, PBCV-1, which replicates in a chlorella-like alga☆

PBCV-1, a large dsDNA-containing virus which replicates in a Chlorella-like green alga, is composed of approximately 64% protein, 25% DNA, and 5-10% lipid on a weight basis. Polyacrylamide gel electrophoresis of the dissociated virus particle resolves 50 to 60 proteins which range in apparent molecular weight from 10,000 to 135,000. Two of these proteins are glycoproteins and at least four are located on the viral surface. The major lipids are phosphatidyl choline, phosphatidyl ethanolamine, and an unidentified component. The effect of organic solvents, surfactants, and chelating and reducing agents on viral infectivity and ultrastructure are reported. Inhibitor studies established that PBCV-1 protein synthesis occurs on cytoplasmic ribosomes.

Properties of the chlorella receptor for the virus PBCV-11

The virus PBCV-1 attached rapidly, specifically, and irreversibly to the external surface of cell walls of its host, a unicellular, eukaryotic Chlorella-like green alga. Attachment was pH and salt dependent. Each cell contained at least 5 X 10(4) PBCV-1 binding sites and Scatchard analysis indicated that each cell could adsorb 5000 PBCV-1 particles. The PBCV-1 receptor was unaffected by extraction with organic solvents, detergents, high salts, or treatment with several proteases as well as the polysaccharide degrading enzymes, cellulase and pectinase. In contrast, acid and alkali treatments of walls at high temperatures and treatment with an enzyme preparation from PBCV-1 lysates destroyed the virus receptor. We suspect that the receptor is a carbohydrate.

A comparison of viruses infecting two different Chlorella-like green Algae☆

Five plaque-forming viruses (Pbi viruses) of the unicellular, eukaryotic, exsymbiotic Chlorella-like green alga strain Pbi were isolated from fresh water collected in Germany. The viruses were compared to two previously characterized plaque-forming viruses (NC64A viruses) of Chlorella strain NC64A. The Pbi viruses do not infect Chlorella NC64A and vice versa. Like the NC64A viruses the Pbi viruses are large polyhedron with a diameter of 140 to 150 nm, are chloroform sensitive, have many structural proteins, and have large dsDNA genomes of at least 300 kb. However, the Pbi viruses are serologically distinct from the NC64A viruses. The five Pbi virus genomes contain 5-methylcytosine, which varied from 14.2 to 43.1% of the cytosine, and two of them also contained N6-methyladenine. DNAs from the Pbi viruses hybridized poorly with the two NC64A virus DNAs and they have a higher guanine plus cytosine content (ca. 46%) than the NC64A virus DNAs (ca. 40%).

Lytic viruses infecting a chlorella-like alga☆

A number of viruses which form plaques on the unicellular, eukaryotic, Chlorella-like green alga, strain NC64A, were isolated from fresh water ponds and rivers in Illinois, North Carolina, and South Carolina. The viruses were large polyhedrons (160 to 190 nm in diameter) and contained dsDNA genomes of ca. 300 kbp. All of the viral DNAs hybridized with DNA from the previously described PBCV-1 virus. However, the viruses, even many of those isolated from the same water sample, could be distinguished from one another by DNA restriction endonuclease digestion. The viruses, including PBCV-1, were grouped into five classes by their resistance to certain DNA restriction endonucleases. Presumably the DNAs in the five classes contain different types or amounts of modified bases.

Infection of a Chlorella-like alga with the virus PBCV-1: Transcriptional studies

Infection of the unicellular, eukaryotic Chlorella-like green alga NC64A by the large dsDNA containing virus PBCV-1 immediately reduced host RNA synthesis. Chloroplast rRNAs, but not cytosolic rRNAs, were degraded following viral infection. Northern blot analysis utilizing four cloned fragments of PBCV-1 DNA as probes, which represent about 12% of the viral genome, revealed several properties of PBCV-1 transcription: A few viral transcripts were detected within 5 min after infection. Each PBCV-1 DNA clone hybridized to both early and late transcripts which implies that early and late genes are dispersed throughout the viral genome. The transition from early to late transcription occurred between 40 and 60 min after infection coincident with the onset of viral DNA synthesis. Three of the four DNA clones hybridized to transcripts which additively were larger than the corresponding DNA probe. This could reflect RNA processing, presence of overlapping genes, or transcription from both DNA strands. A few, but not all, early transcripts were synthesized in the presence of cycloheximide. This suggests that the virus either carries in its own RNA polymerase or uses a host RNA polymerase for very early viral transcription and that synthesis of additional, later transcripts depends on translation of an early gene product(s).