PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, "Pyrococcus abyssi"

Abstract

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "Pyrococcus abyssi" previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the Fuselloviridae, isolated from Sulfolobus shibatae. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of Thermococcales isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.

FIG. 1.

Electron micrographs of PAV1 negatively stained with 2% uranyl acetate. (a) Virus particles attached to cellular material. (b) Purified free virus particles obtained after centrifugation in a CsCl buoyant-density gradient. (c) Elongated virus particles indicating plasticity of form. (d) Effect of exposure to chloroform on virus particle structure. (e) Effect of exposure to proteinase K on virus particle structure. Bars, 100 nm.

FIG. 2.

Agarose gel electrophoresis of the cccDNA from strain GE23 cut by BamHI (lane 2), EcoRI (lane 3), HindIII (lane 4) and PstI (lane 5) and uncut (lane 6). Lane 1, DNA size marker (kb) showing λDNA digested with HindIII, BamHI, and EcoRI.

FIG. 3.

Comparison of cccDNA from strain GE23 with DNA from purified PAV1 VLP. Lanes 1 and 3, plasmid DNA; lanes 2 and 4; VLP DNA. Lanes 1 and 2, digested with HindIII; lanes 3 and 4, digested with PstI. Lane 5, 1-kb DNA ladder.

FIG. 4.

Silver-stained SDS-polyacrylamide gel electrophoresis of the PAV1 proteins. The results for three independent preparations of CsCl-gradient purified VLPs are shown. Lanes 3 and 4 are still contaminated with host proteins (presumably from host flagella); lane 5 contains mostly PAV1 proteins indicated by an arrow. Lanes 1 and 2, Kaleidoscope prestained standards and unstained Precision protein standards (Bio-Rad); the subunit sizes are listed in kDa at the left of the figure.

FIG. 5.

Strain GE23 growth curve (cells/ml) (•) and time course of VLP production (⧫). The approximate titer of free VLP was estimated by a standardized electron microscopic count together with the corresponding host cell count.

FIG. 6.

Southern hybridization analysis. Total DNA and cccDNA from the strain GE23 were digested with HindIII (lanes 1 and 2, respectively) or PstI (lanes 3 and 4, respectively) and separated on agarose gels (0.8%) before blotting. The pooled HindIII fragments of the cccDNA, labeled with fluorescein-11-dUTP, were used as a probe.