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. 2012 Jul;40(12):5523-34.
doi: 10.1093/nar/gks215. Epub 2012 Mar 6.

Related haloarchaeal pleomorphic viruses contain different genome types

Ana Sencilo  1 , Lars Paulin, Stefanie Kellner, Mark Helm, Elina Roine
Affiliations

Related haloarchaeal pleomorphic viruses contain different genome types

Ana Sencilo et al. Nucleic Acids Res. 2012 Jul.

Abstract

Archaeal viruses have been the subject of recent interest due to the diversity discovered in their virion architectures. Recently, a new group of haloarchaeal pleomorphic viruses has been discovered. It is distinctive in terms of the virion morphology and different genome types (ssDNA/dsDNA) harboured by rather closely related representatives. To date there are seven isolated viruses belonging to this group. Most of these share a cluster of five conserved genes, two of which encode major structural proteins. Putative proviruses and proviral remnants containing homologues of the conserved gene cluster were also identified suggesting a long-standing relationship of these viruses with their hosts. Comparative genomic analysis revealed three different ways of the genome organization, which possibly reflect different replication strategies employed by these viruses. The dsDNA genomes of two of these viruses were shown to contain single-strand interruptions. Further studies on one of the genomes suggested that the interruptions are located along the genome in a sequence-specific manner and exhibit polarity in distribution.

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Figures

Figure 1.
Figure 1.
Schematic genomic alignment of the haloarchaeal pleomorphic viruses HHPV-1, HRPV-6, HRPV-2, HRPV-1, HRPV-3 and HGPV-1 as linear representation and His2 (5) (GenBank accession no AF191797). HRPV-1 genome is used as a reference. Genes encoding VP3-like, VP4-like and VP8-like protein homologues are marked below the alignment of the genomes. Genes encoding structural proteins of HGPV-1 and His2 determined by M.K. Pietilä et al. (21) are denoted by asterisk. Percentages of identical amino acids in homologous putative polypeptides and proteins are indicated. Subgroups to which viruses were assigned according to genome organization are indicated on the right.
Figure 2.
Figure 2.
Mung bean nuclease (MBN) analyses of HRPV-3 and HGPV-1 genomes. (A) Digestion of HRPV-3 and HGPV-1 genomes using concentrations of either 0.5 or 5 units (U) of MBN per 1 µg of genomic DNA. Virion φX174 DNA (ssDNA) and its replicative form RFII (dsDNA) were used as controls. (B) MBN digestions (0.5 U of MBN/1 µg of genomic DNA); the genomes were treated with either T4 DNA ligase (lig.) or Sulfolobus DNA polymerase IV and T4 DNA ligase (PolIV lig.). GeneRulerTM DNA ladder mix was used as a standard (‘st.’).
Figure 3.
Figure 3.
Mapping of the discontinuous sites in the HRPV-3 genome. (A) Conserved nucleotides of the aligned sequences showing the identified motif found in the MBN-resistant fragment ends of HRPV-3. (B) Sanger sequencing of the genomic site with predicted discontinuity. A drop in sequencing signal was observed after the motif GCCCA when HRPV-3 genome was used as a template (7502 F_genome). Sequencing of corresponding HRPV-3 fragment amplified by PCR (7502 F_PCR) showed no decrease in the signal. (C) HRPV-3 genome map with arrows marking predicted sites of discontinuities (GCCCA motif). The names of primers used to sequence the regions with putative discontinuities are noted above or below the arrow. Sites which were not possible to sequence because of the proximity of the other interruption are marked with grey arrow. Black arrows indicate the predicted sites of discontinuity where a sudden drop of sequencing signal was observed as exemplified by the above-placed sequencing chromatograph (7502F_genome). Red arrows indicate positions where no sequencing signal drop was detected despite the predicted motif for discontinuity.
Figure 4.
Figure 4.
LC-MS/MS analysis of HGPV-1 and HRPV-3 digests. (A) MRM chromatograms of a DNA digest, RNA digest including 5-deoxymethylcytidine, HGPV-1 DNA digest and HRPV-3 DNA digest. (B) Reactions monitored by MRM and retention times of nucleosides. (C) UV traces at 254 nm of HGPV-1 DNA digest and HRPV-3 DNA digest used for semi-quantitative calculations.
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