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. 2018 Feb 28;9(3):131.
doi: 10.3390/genes9030131.

The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules

Nina S Atanasova  1   2 , Camilla H Heiniö  3 , Tatiana A Demina  4 , Dennis H Bamford  5 , Hanna M Oksanen  6
Affiliations

The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules

Nina S Atanasova et al. Genes (Basel). .

Abstract

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.

Keywords: Pleolipoviridae; genomic diversity; haloarchaeal virus; hypersaline environment; pleolipovirus; pleomorphic virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Haloarcula hispanica pleomorphic virus 4 (HHPV4) infectivity in different ionic conditions. (a) Infectivity in HHPV4 buffer (3.4 M NaCl, 120 mM MgSO4, 110 mM MgCl2, 70 mM KCl, 4 mM CaCl2, 50 mM Tris-HCl, pH 7.2) from which one or two components at a time have been removed. Infectivity was assayed after 3 h (black bars) and 24 h (grey bars) incubation at 4 °C. Modified growth medium (MGM) and agar stock were used as controls; (b) The effect of CaCl2 concentration in HHPV4 buffer on virus infectivity after 3 h (circles) and 24 h (triangles) incubation at 4 °C. Chelating agent EGTA was used to remove residual CaCl2; (c) The effect of NaCl concentration in HHPV4 buffer (pH 7.2) on virus infectivity after 7 d incubation at 4 °C (circles). Virus infectivity in HHPV4 buffer at pH 6.2 (crosses) and pH 9.1 (triangles) in the presence of 1 M and 3.4 M NaCl were assayed in the same incubation conditions. Pfu, plaque-forming unit.
Figure 2
Figure 2
Virus adsorption and life cycle. (a) Adsorption of HHPV4 to Haloarcula hispanica cells at 37 °C. Error bars represent one standard deviation (n = 3); (b–d) Life cycle of HHPV4 in H. hispanica; (b) Turbidity of the infected (black circles) and uninfected (white circles) H. hispanica cultures and the number of free infectious viruses (grey bars); (c) Viable cells of the infected (black circles) and uninfected (white circles) cultures; (d) Infective centres of the infected H. hispanica culture for the first 2.5 h post infection (p.i.). Error bars represent one standard deviation in (c) and (d) (n = 3). Cfu, colony-forming unit.
Figure 3
Figure 3
HHPV4 purification and structural proteins. (a) Infectivity (grey bars), absorbance (black circles), and density (g/mL; open circles) of the 20–60% (w/v) sucrose gradient fractions (2× purified HHPV4 particles). Fraction numbers correspond to 4 mL fractions (AH629 tube). No. 1 is the top of the gradient. The position of the light scattering zone with the highest infectivity is indicated by a horizontal segment of a line; (b) The protein pattern of the virus peak analysed by SDS-PAGE and Coomassie Blue staining (the position of the segment of a line in a). (c) Comparison of the protein profiles of the 1× and 2× purified HHPV4 to that of the 1× purified HHPV3 (Coomassie Blue stained SDS-PAGE gel). HHPV4 proteins (virion proteins, VP) VP9 and VP11, as well as the corresponding HHPV3 proteins VP1 and VP3 [4], are indicated.
Figure 4
Figure 4
HHPV4 virion and its lipids. (a) Transmission electron microscopy (TEM) micrograph of the 2× purified HHPV4 particles negatively stained with 1% ammonium molybdate. Scale bar equals 100 nm; (b) Thin-layer chromatogram of lipids extracted from H. hispanica cells and 2× purified HHPV4 particles. The major lipid species of H. hispanica [55] are indicated on the left: PG, phosphatidylglycerol; PGP-Me, phosphatidylglycerophosphate methyl ester; PGS, phosphatidylglycerosulfate; TGD, triglycosyl glycerodiether.
Figure 5
Figure 5
Comparison of genomes of betapleolipoviruses. All genomes are circular double stranded (ds) DNA molecules, which are shown as linearized molecules. Open reading frames (ORFs) or genes are represented as grey arrows and are numbered (gene numbers are italicized). GenBank accession numbers are indicated in brackets, and the total length of genome sequences is marked under virus name. Nucleotide coordinates are shown in purple. (a) Comparison of HHPV4 and HHPV3 genomes. The regions of 100% nucleotide identity are marked with blue, and nucleotide coordinates of the identical regions are shown in dark green; (b) All known betapleolipovirus genomes are compared. Virion proteins (VPs) are marked. Similar ORFs or genes are in the same colours. Amino acid identities (%) between (putative) proteins are shown in between the genomes. HRPV-3, Halorubrum pleomorphic virus 3; HGPV-1, Halogeometricum pleomorphic virus 1.
Figure 6
Figure 6
Phylogenomic Genome BLAST Distance Phylogeny (GBDP) trees of the whole genomic sequences of the members and putative members of the family Pleolipoviridae at the (a) nucleotide and (b) amino acid levels. Viruses classified or proposed to be classified (marked by asterisks) into three genera of the family Pleolipoviridae are marked with different colours (see the colour code). Genera yielded by OPTSIL clustering are shown as squares. The branch lengths are scaled in terms of the GBDP distance formula D0 in (a) and the GBDP distance formula D6 in (b) [49]. The trees and OPTSIL clusters were obtained using the VICTOR web service [49].
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