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Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.

We have sequenced the genome and identified the structural proteins and lipids of the novel membranecontaining, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an 17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Haloarcula provirus), and the Haloarcula virus SH1. These genetic elements share two major capsid proteins and a putative packaging ATPase. The ATPase is similar with the ATPases found in the PRD1-type viruses, thus providing an evolutionary link to these viruses and furthering our knowledge on the origin of viruses. Three-dimensional structures of the major capsid proteins, as well as the architecture of the virion and the sequence similarity of putative genome packaging ATPases, have revealed unexpected evolutionary connection between virus families. Viruses infecting hosts residing in different domains of life (Bacteria, Archaea, and Eukarya) share common structural elements and possibly also ways to package the viral genome (8, 13, 41). It has been proposed that the set of genes responsible for virion assembly is a hallmark of the virus and is designated as the innate viral “self,” which may retain its identity through evolutionary times (5). Based on this, it is proposed that viruses can be classified into lineages that span the different domains of life. Therefore, the studies of new virus isolates might provide insights into the events that led to the origin of viruses and maybe even the origin of life itself (34, 40). However, viruses are known to be genetic mosaics (28), and these structural lineages therefore do not reflect the evolutionary history of all genes in a given virus. For example, the genome replication strategies vary significantly even in the currently established lineages (41) and, consequently, a structural approach does not point out to a specific form of replication in the ancestor. Nevertheless, as the proposal for a viral self is driven from information on viral structures and pathways of genome encapsidation, the ancestral form of the self was likely to be composed of a protective coat and the necessary mechanisms to incorporate the genetic material within the coat. Viruses structurally related to bacteriophage PRD1, a phage infecting gram-negative bacteria, have been identified in all three domains of life, and the lineage hypothesis was first proposed based on structural information on such viruses. Initially, PRD1 and human adenovirus were proposed to originate from a common ancestor mainly due to the same capsid organization (T25)

Viruses infecting archaeal cells are less well understood than those infecting eukaryotic and bacterial cells. Here we study the distribution of the structural proteins between the capsid and the membrane of icosahedral SH1 virus, an archaeal virus infecting extreme halophilic Haloarcula hispanica cells. General features such as morphology, linear dsDNA genome and presence of lipids suggest that it may belong to the recently proposed PRD1-adenovirus lineage of viruses. To investigate this we have initiated structural studies of the virion. Quantitative dissociation of SH1 by 3 M urea or by lowering the salt concentration identified a number of soluble capsid-associated proteins (VP2, VP3, VP4, VP6, VP7 and VP9). These released proteins left behind a particle, or lipid core, containing two major proteins VP10 and VP12 and viral phospholipids. VP1 was released from the lipid core in low ionic strength conditions but not with 3 M urea. Approximately half of the protein VP5 stayed with the lipid core and the other half was released. Analysis of the soluble capsid-associated proteins by their sedimentation and hydrodynamic properties suggests that the most abundant proteins, putative capsomers VP4 and VP7, form an intricate pattern of protein complexes. We also observed large differences in the sizes of the complexes determined by the two different methods suggesting an elongated overall structure for most of the capsid-associated proteins or protein complexes. This work verifies that there is an internal membrane vesicle residing inside the complex icosahedral capsid that is akin to the overall structure of PRD1-like viruses.

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.

The phospholipid (PL) molecular species compositions of bacteriophages PRD1 and Bam35 as well as their respective hosts were determined quantitatively using liquid chromatography/electrospray ionization mass-spectrometry (LC-ESI-MS) and backed up by gas-chromatographic/mass-spectrometric (GC-MS) analysis of the total fatty acids (FAs). The results showed that both viruses contain significantly more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membranes. Only modest differences in the molecular species composition of the viruses and their respective hosts were observed, indicating that the virus assembly process is relatively nonselective in respect of the fatty acid (FA) proportion of phospholipids (PL). These data indicate that the PL composition of these two viruses is largely, albeit not exclusively, determined by the availability of phospholipids in the host membrane.