photosynthesis, cell cycle regulation, cell motility, and non-Mendelian (chloroplast) inheritance. Given the importance of eukaryotic algae, there is surprisingly little information about viruses or viruslike particles (VLP) in these organisms. It is difficult to credit the first person who described a virus or VLP in a eukaryotic alga. The problem is complicated because early reports consisted solely of microscopic observations and, in some cases, cultures were not axenic. A few papers in the Russian literature as long as 30 years ago described a lytic activity in cultures of the green alga Chlorella pyrenoidosa (174, 224-226). This lytic activity was given the name "chlorellophage." However, these investigators acknowledged that their cultures were contaminated with bacteria. Furthermore, the chlorellophage had typical bacteriophage morphology (174, 224). Therefore, despite the name chlorellophage, the host for these particles is unclear. Several investigators, almost simultaneously, reported the presence of VLPs in eukaryotic algae in the early 1970s. Lee (73) described 50to 60-nm polyhedral particles in vegetative cells of the red alga Sirodotia tenuissima, Pickett-Heaps 586

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses.

The 331-kbp chlorovirusParamecium bursariachlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its hostChlorella variabilisNC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

Four spontaneously derived serologicafly distinct classes of mutants of the Paramecium bursaria chlorella virus (PBCV-1) were isolated using polydonal antiserum prepared against either intact PBCV-l or PBCV-1-derived sero-types. The oligosaccharide(s) of the viral major capsid protein and two minor glycoproteins determined virus serological specificity. Normally, viral glycoproteins arise from host-specific glycosylation of viral proteins; the glycan portion can be altered only by growing the virus on another host or by mutations in glycosylation sites of the viral protein. Neither mechanism explains the changes in the glycan(s) ofthe PBCV-1 maJor capsid protein because all of the viruses were grown in the same host alga and the predicted amino acid sequence of the major capsid protein was identical in the PBCV-1 serotypes. PBCV-1 antiserum resistance is best explained by viral mutations that block specific steps in glycosylation, possibly by inactivating glycosyltansferases.Paramecium bursaria chlorella virus (PBCV-1) is a large (==190 nm in diameter) polyhedral plaque-forming virus that replicates in certain unicellular eukaryotic exsymbiotic Chlorella-like green algae (1). The PBCV-1 genome is a linear nonpermuted 333-kb double-stranded DNA with covalently closed hairpin ends (2, 3). PBCV-1 contains at least 50 proteins and a lipid component located inside the capsid shell (4). The major capsid protein (Vp54) ofPBCV-1 is one offour proteins located on the viral surface and is one of three glycosylated viral proteins. PBCV-1 and its related viruses have recently been assigned to a virus family, named Phycodnaviridae (5). Additional features of these viruses have been reviewed (6).Chase et al. (7) showed that chlorella viruses exclude one another during dual inoculation of the host. For these studies we isolated a spontaneous mutant of PBCV-1, named EPA-1, which was resistant to PBCV-1 polyclonal antiserum (dilution of 1:4). This antiserum completely inhibited PBCV-1 infection even at a dilution of 1:1000. Polyclonal antiserum prepared against EPA-1 inhibited EPA-1 infection (dilution of 1:1000) but not PBCV-1 infection (dilution of 1:4). In this study we have addressed two questions: (i) Can additional PBCV-1 serotypes be isolated using polyclonal antisera to intact viruses? (ii) What virus component determines antiserum resistance? We have found four PBCV-1 serotypes and discovered that the antisera react primarily with oligosaccharide(s) attached to the major capsid protein plus two minor glycoproteins. More importantly, in contrast to normal virus protein glycosylation, the virus rather than the host dictates glycosylation specificity.MATERIALS AND METHODS Culture Conditions. The procedures for producing, purifying, and "plaquing" the viruses and the growth ofthe host alga (Chlorella NC64A) on MBBM medium have been described (1,8). Virus glycoproteins were specifically labeled by adding 100 ,uCi of D-[6-3H]-galactose (1 Ci = 37 GBq) to 1 ml of cells (6 x 108 cells per ml) 30 min after virus infectio...

Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D 2 O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N-and Cterminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids.