The Closest Relatives of Icosahedral Viruses of Thermophilic Bacteria Are among Viruses and Plasmids of the Halophilic Archaea

We have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, 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 (T=25) and the major coat protein topology, the trimeric double β-barrel fold (12). In addition, these viruses share a common vertex organization and replication mechanism (20, 31, 53, 63). PRD1 is an icosahedral virus with an inner membrane, whereas adenovirus lacks the membrane. Later, many viruses with similar double β-barrel fold in the major coat protein have been discovered and included to this viral lineage. For example, the fold is present in Paramecium bursaria Chlorella virus 1 (56) of algae, Bam35 (45) of gram-positive bacteria, PM2 (2) of gram-negative marine bacteria, and Sulfolobus turreted icosahedral virus (STIV) (38) of an archaeal host. Moreover, genomic analyses have revealed a common set of genes in a number of nucleocytoplasmic large DNA viruses. Chilo iridescent virus and African swine fever virus 1 are related to Paramecium bursaria Chlorella virus 1 and most probably share structural similarity to PRD1-type viruses (13, 30, 31, 68). The largest known viruses, represented by mimivirus and poxvirus, may also belong to this lineage (29, 77). Two euryarchaeal proviruses, TKV4 and MVV, are also proposed to belong to this lineage based on bioinformatic searches (42). The proposed PRD1-related viruses share the same basic architectural principles despite major differences in the host organisms and particle and genome sizes (1, 2, 38, 56). PM2, for example, has a genome of only 10 kbp, whereas mimivirus (infecting Acanthamoeba polyphaga) double-stranded DNA (dsDNA) genome is 1.2 Mbp in size (59).How many virion structure-based lineages might there be? This obviously relates to the number of protein folds that have the properties needed to make viral capsids. It has been noted that, in addition to PRD1-type viruses, at least tailed bacterial and archaeal viruses, as well as herpesviruses, share the same coat protein fold. Also, certain dsRNA viruses seem to have structural and functional similarities, although their hosts include bacteria and yeasts, as well as plants and animals (6, 18, 19, 27, 55, 60, 74). Obviously, many structural principles to build a virus capsid exist, and it has been suggested that especially geothermally heated environments have preserved many of the anciently formed virus morphotypes (35).Thermophilic dsDNA bacteriophage P23-77 was isolated from an alkaline hot spring in New Zealand on Thermus thermophilus (17) ATCC 33923 (deposited as Thermus flavus). P23-77 was shown to have an icosahedral capsid and possibly an internal membrane but no tail (81). Previously, another Thermus virus, IN93, with a similar morphology has been described (50). IN93 was inducible from a lysogenic strain of Thermus aquaticus TZ2, which was isolated from hot spring soil in Japan. Recently, P23-77 was characterized in more detail (33). It has an icosahedral protein coat, organized in a T=28 capsid lattice (21). The presence of an internal membrane was confirmed, and lipids were shown to be constituents of the virion. Ten structural proteins were identified, with apparent molecular masses ranging from 8 to 35 kDa. Two major protein species with molecular masses of 20 and 35 kDa were proposed to make the capsomers, one forming the hexagonal building blocks and the other the two towers that decorate the capsomer bases (33). Surprisingly, P23-77 is structurally closest to the haloarchaeal virus SH1, which is the only other example of a T=28 virion architecture (32, 33). In both cases it was proposed that the capsomers are made of six single β-barrels opposing the situation with the other structurally related viruses where the hexagonal capsomers are made of three double β-barrel coat protein monomers (8).In the present study we analyze the dsDNA genome of P23-77. Viral membrane proteins and those associated with the capsid were identified by virion dissociation studies. The protein chemistry data and genome annotation are consistent with the results of the disruption studies. A detailed analysis of the lipid composition of P23-77 and its T. thermophilus host was carried out. The data collected here reveal additional challenges in attempts to generate viral lineages based on the structural and architectural properties of the virion.     

Adaptations to fasting in the American mink (<Emphasis Type="Italic">Mustela vison</Emphasis>): nitrogen metabolism

The aim of this study was to investigate the adaptations of protein metabolism to seasonal fasting in an actively wintering boreal carnivore. Fifty farm-bred male American minks Mustela vison were divided into a fed control group and four experimental groups fasted for 2, 3, 5 or 7 days. The responses of nitrogen metabolism to wintertime food deprivation were determined by measuring the rate of weight loss, the tissue total protein concentrations and the plasma amino acid, urea, ammonia, uric acid and total protein levels. The mink has relatively poor adaptations to food deprivation, as it is not able to prolong phase II of fasting with fat as the major metabolic fuel. Instead, the species has to derive a part of its energy requirements from the breakdown of body proteins. The end product of protein catabolism—urea— accumulates in its circulation, and the mink may not be able to recycle urea-N. Although the mink can still have a high body fat percent at the end of the 7-day fast, it appears to enter phase III of fasting with stimulated proteolysis during this period.     

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [³H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [³H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.     

Environmental variability and population dynamics: do European and North American ducks play by the same rules?

Density dependence, population regulation, and variability in population size are fundamental population processes, the manifestation and interrelationships of which are affected by environmental variability. However, there are surprisingly few empirical studies that distinguish the effect of environmental variability from the effects of population processes. We took advantage of a unique system, in which populations of the same duck species or close ecological counterparts live in highly variable (north American prairies) and in stable (north European lakes) environments, to distinguish the relative contributions of environmental variability (measured as between‐year fluctuations in wetland numbers) and intraspecific interactions (density dependence) in driving population dynamics. We tested whether populations living in stable environments (in northern Europe) were more strongly governed by density dependence than populations living in variable environments (in North America). We also addressed whether relative population dynamical responses to environmental variability versus density corresponded to differences in life history strategies between dabbling (relatively “fast species” and governed by environmental variability) and diving (relatively “slow species” and governed by density) ducks. As expected, the variance component of population fluctuations caused by changes in breeding environments was greater in North America than in Europe. Contrary to expectations, however, populations in more stable environments were not less variable nor clearly more strongly density dependent than populations in highly variable environments. Also, contrary to expectations, populations of diving ducks were neither more stable nor stronger density dependent than populations of dabbling ducks, and the effect of environmental variability on population dynamics was greater in diving than in dabbling ducks. In general, irrespective of continent and species life history, environmental variability contributed more to variation in species abundances than did density. Our findings underscore the need for more studies on populations of the same species in different environments to verify the generality of current explanations about population dynamics and its association with species life history.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

Effects of Auditory Attention Training with the Dichotic Listening Task: Behavioural and Neurophysiological Evidence

Facilitation of general cognitive capacities such as executive functions through training has stirred considerable research interest during the last decade. Recently we demonstrated that training of auditory attention with forced attention dichotic listening not only facilitated that performance but also generalized to an untrained attentional task. In the present study, 13 participants underwent a 4-week dichotic listening training programme with instructions to report syllables presented to the left ear (FL training group). Another group (n = 13) was trained using the non-forced instruction, asked to report whichever syllable they heard the best (NF training group). The study aimed to replicate our previous behavioural results, and to explore the neurophysiological correlates of training through event-related brain potentials (ERPs). We partially replicated our previous behavioural training effects, as the FL training group tended to show more allocation of auditory spatial attention to the left ear in a standard dichotic listening task. ERP measures showed diminished N1 and enhanced P2 responses to dichotic stimuli after training in both groups, interpreted as improvement in early perceptual processing of the stimuli. Additionally, enhanced anterior N2 amplitudes were found after training, with relatively larger changes in the FL training group in the forced-left condition, suggesting improved top-down control on the trained task. These results show that top-down cognitive training can modulate the left-right allocation of auditory spatial attention, accompanied by a change in an evoked brain potential related to cognitive control.     

The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.