In the early 1980s, Dr. Meints and Dr. Van Etten were studying Chlorella-like green algae that live in a symbiotic relationship within cells of the protozoan Hydra viridis. They found that the algal cells could be excised from the hydra, which could exist free of the symbiont if given proper nutrients. However, it was not possible to culture the algae free of the hydra host. Further study demonstrated that when the algal cells were isolated from the host, virus particles rapidly began to multiply within the algae, resulting in lysis of the algal population within 24 hours. Ultrastructual and biochemical studies on this algal virus system produced the following results:

• The virus consisted of a large (approximately 190-nm), polygonal particle, containing 30 to 40 polypeptides, the most abundant of which was a 46 kDa glycoprotein, presumably associated with the viral capsid.

• The virus genome consists of about 130 kbp of double-stranded DNA.

• New virus particles were assembled in the cytoplasm of the algal cells and released upon lysis of the algal cell wall.

This virus, called HVCV (for Hydra viridis Chlorella virus), was one of the few viruses described in eukaryotic algae. HV CV might play a role in initiating or maintaining the symbiotic

relationship between the alga and its hydra host, possibly by altering the algal cell wall. Subsequent studies identified viruses in four other strains of Hydra obtained from commercial sources. The viruses fell into two classes, based on particle size, bouyant density, and DNA restriction patterns (HV CV -1, HV CV -2). In addition, a similar virus was isolated from symbiotic Chlorella from Paramecium bursaria (PBCV-1).

To facilitate the study of these viruses, it was desirable to identify an algal strain that could be cultured free of the hydra or Paramecium host, which was susceptible to infection by the virus. This would allow production of large quantities of the virus and the study of viral replication and development. Sixteen strains of culturable Chlorella, which had been isolated from invertebrates such as Paramecium, Hydra, Stentor, and sponges, plus two free-living strains, were obtained. Attempts were made to infect these Chlorella strains with all the virus strains described earlier. None of the HV CV viruses (from Chlorella-Hydra hosts) were able to infect any Chlorella strain tested. However, two culturable Chlorella strains originally isolated from Paramecium (Chlorella strains N1a and NC64) were infected with PBCV-1 (the P. bursaia Chlorella virus). The infection led to lysis of the algal cells and production of large amounts of infectious viral progeny. This result led to the development of a plaque assay system for the algal viruses, similar to a bacteriophage assay on bacterial lawns. The availability of this system, which caused synchronous infection of the algal cells and the production of large quantities of viral particles, allowed the researchers to characterize the virus biochemically. It also allowed researchers to study the regulation of viral gene expression and the effects of viral replication on algal physiology and gene expression. A large number of publications resulted from this research (see below). Several of the most interesting and possibly relevant findings are summarized here.

• The virus particles attach at one vertex of their polygonal capsid to receptor sites on the algal cell wall. A lytic enzyme produced by the virus degrades the wall at this site, and the viral DNA is released into the cell. Living algal cells are not required for virus attachment and wall degradation (viruses can attach to and degrade isolated wall fragments), but living cells are necessary for release of the viral DNA. Complete viral capsids are assembled in viral assembly sites within the cytoplasm and subsequently filled with DNA. Virus particles are released through holes produced at discrete locations in the algal cell wall.

• The plaque assay system was used to screen for other virues that infect algae.

Viruses that infect Chlorella strains N1a or NC64A were found to be very common in nature. The viruses all had similar features, including a large, polygonal capsid and dsDNA; however, some viruses were distinguishable based on plaque size, reactivity to anti-PBCV -1 antisera, variations in the DNA restriction patterns and the extent of nucleotide modification. [7]

expression of overlapping genes or transcription of genes from both DNA strands. Analysis of DNA from some viral isolates showed that the viral DNA is modified to varying extents, primarily in the methylation of adenine and cytosine residues. The data suggested that the virus produces a unique restriction enzyme that is specific for non-methylated sequences for degradation of the host DNA. The virus also produces a corresponding methyltransferase, which recognizes the same sequence as the restriction endonuclease. The methyltransferase methylates newly synthesized viral DNA, protecting it from degradation in the next round of infection.

Dr. Meints received funding from SERI from 1986 through 1989. The overall goal of the SERI — funded research was to use the algal virus system to develop methods for genetically manipulating microalgae with potential for liquid fuel production. The research from Dr. Meints’ laboratory is reviewed below with respect to the specific goals of the project.