Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica

Abstract

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.

FIG. 1.

(A) Thin-layer chromatogram of the lipids extracted from H. hispanica and SH1. Lipids were identified on the basis of their relative retention times (which were very similar to previously reported ones [21]), staining behavior, and their molecular mass as determined by MS. Some lipids present in the host lipid extract could not be identified. Due to the limited amount of virus available, the amount of total lipids on the SH1 lane is somewhat less than the amount on the host lane. (B) Negative ion mass spectrum of the lipids extracted from H. hispanica (upper panel) and SH1 (lower panel). Inset in the upper panel is a magnification of the 1,120 to 1,200 m/z region from the spectrum of the host lipid extract showing TGD and its adduct. Peaks representing doubly charged noncovalent lipid heterodimers are marked with an asterisk (*). For example, m/z 910.9 is a doubly charged dimer of PGP-Me (m/z 899.5) and PGP-Me plus Na (m/z 921.4), whereas m/z 1019.2 is a doubly charged dimer of PGP-Me (m/z 899.5) and TGD (m/z 1137.6). NL, neutral lipids; CL, cardiolipin.

FIG. 2.

Genome organization of SH1. (A) Predicted ORFs are numbered 1 to 56 from left to right and shaded according to the calculated isoelectric point of the predicted gene products. Structural virion components (VP1 to VP14) determined by protein chemistry methods are marked. (B) Direction of transcription is depicted by arrows. (C) Scale bar.

FIG. 3.

SDS-PAGE (17% acrylamide) of the purified SH1 depicting the structural proteins (lane 2). Molecular mass marker are shown in lane 1.

FIG. 4.

Schematic presentation of VP2. The two linker-type regions (11G residues and GS-rich sequence) divide the protein in three domains. The repeated sequences in the N- and C-terminal domains are shown below.

FIG. 5.

Nonreducing SDS-PAGE (8% acrylamide) of SH1 showing proteins VP1 and VP2 and protein complexes from 50 kDa to several hundred kDa (C1 to C5). Molecular mass markers (kDa) are shown on the left. The bands were further subjected to peptide mass fingerprinting to identify their protein composition (see text for details).