Gisle Vestergaard

Virology: Independent virus development outside a host

Viruses are thought to be functionally inactive once they are outside and independent of their host cell. Here we describe an exceptional property of a newly discovered virus that infects a hyperthermophilic archaeon growing in acidic hot springs: the lemon-shaped viral particle develops a very long tail at each of its pointed ends after being released from its host cell. The process occurs only at the temperature of the hosts habitat (75–90 °C) and it does not require the presence of the host cell, an exogenous energy source or any cofactors. This host-independent morphological development may be a strategy for viral survival in an environment that is unusually harsh and has limited host availability.

CRISPR adaptive immune systems of Archaea

CRISPR adaptive immune systems were analyzed for all available completed genomes of archaea, which included representatives of each of the main archaeal phyla. Initially, all proteins encoded within, and proximal to, CRISPR-cas loci were clustered and analyzed using a profile–profile approach. Then cas genes were assigned to gene cassettes and to functional modules for adaptation and interference. CRISPR systems were then classified primarily on the basis of their concatenated Cas protein sequences and gene synteny of the interference modules. With few exceptions, they could be assigned to the universal Type I or Type III systems. For Type I, subtypes I-A, I-B, and I-D dominate but the data support the division of subtype I-B into two subtypes, designated I-B and I-G. About 70% of the Type III systems fall into the universal subtypes III-A and III-B but the remainder, some of which are phyla-specific, diverge significantly in Cas protein sequences, and/or gene synteny, and they are classified separately. Furthermore, a few CRISPR systems that could not be assigned to Type I or Type III are categorized as variant systems. Criteria are presented for assigning newly sequenced archaeal CRISPR systems to the different subtypes. Several accessory proteins were identified that show a specific gene linkage, especially to Type III interference modules, and these may be cofunctional with the CRISPR systems. Evidence is presented for extensive exchange having occurred between adaptation and interference modules of different archaeal CRISPR systems, indicating the wide compatibility of the functionally diverse interference complexes with the relatively conserved adaptation modules.

Archaeal CRISPR-based immune systems: exchangeable functional modules

CRISPR (clustered regularly interspaced short palindromic repeats)-based immune systems are essentially modular with three primary functions: the excision and integration of new spacers, the processing of CRISPR transcripts to yield mature CRISPR RNAs (crRNAs), and the targeting and cleavage of foreign nucleic acid. The primary target appears to be the DNA of foreign genetic elements, but the CRISPR/Cmr system that is widespread amongst archaea also specifically targets and cleaves RNA in vitro. The archaeal CRISPR systems tend to be both diverse and complex. Here we examine evidence for exchange of functional modules between archaeal systems that is likely to contribute to their diversity, particularly of their nucleic acid targeting and cleavage functions. The molecular constraints that limit such exchange are considered. We also summarize mechanisms underlying the dynamic nature of CRISPR loci and the evidence for intergenomic exchange of CRISPR systems.

Stygiolobus Rod-Shaped Virus and the Interplay of Crenarchaeal Rudiviruses with the CRISPR Antiviral System

A newly characterized archaeal rudivirus Stygiolobus rod-shaped virus (SRV), which infects a hyperthermophilic Stygiolobus species, was isolated from a hot spring in the Azores, Portugal. Its virions are rod-shaped, 702 (+/- 50) by 22 (+/- 3) nm in size, and nonenveloped and carry three tail fibers at each terminus. The linear double-stranded DNA genome contains 28,096 bp and an inverted terminal repeat of 1,030 bp. The SRV shows morphological and genomic similarities to the other characterized rudiviruses Sulfolobus rod-shaped virus 1 (SIRV1), SIRV2, and Acidianus rod-shaped virus 1, isolated from hot acidic springs of Iceland and Italy. The single major rudiviral structural protein is shown to generate long tubular structures in vitro of similar dimensions to those of the virion, and we estimate that the virion constitutes a single, superhelical, double-stranded DNA embedded into such a protein structure. Three additional minor conserved structural proteins are also identified. Ubiquitous rudiviral proteins with assigned functions include glycosyl transferases and a S-adenosylmethionine-dependent methyltransferase, as well as a Holliday junction resolvase, a transcriptionally coupled helicase and nuclease implicated in DNA replication. Analysis of matches between known crenarchaeal chromosomal CRISPR spacer sequences, implicated in a viral defense system, and rudiviral genomes revealed that about 10% of the 3,042 unique acidothermophile spacers yield significant matches to rudiviral genomes, with a bias to highly conserved protein genes, consistent with the widespread presence of rudiviruses in hot acidophilic environments. We propose that the 12-bp indels which are commonly found in conserved rudiviral protein genes may be generated as a reaction to the presence of the host CRISPR defense system.

Structure and Genome Organization of AFV2, a Novel Archaeal Lipothrixvirus with Unusual Terminal and Core Structures

A novel filamentous virus, AFV2, from the hyperthermophilic archaeal genus Acidianus shows structural similarity to lipothrixviruses but differs from them in its unusual terminal and core structures. The double-stranded DNA genome contains 31,787 bp and carries eight open reading frames homologous to those of other lipothrixviruses, a single tRNA(Lys) gene containing a 12-bp archaeal intron, and a 1,008-bp repeat-rich region near the center of the genome.

CRISPR-based immune systems of the Sulfolobales: complexity and diversity

CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems are presented.

Structure of the Acidianus Filamentous Virus 3 and Comparative Genomics of Related Archaeal Lipothrixviruses

ABSTRACT Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-μm-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters.

Independent virus development outside a host

Viruses are thought to be functionally inactive once they are outside and independent of their host cell. Here we describe an exceptional property of a newly discovered virus that infects a hyperthermophilic archaeon growing in acidic hot springs: the lemon-shaped viral particle develops a very long tail at each of its pointed ends after being released from its host cell. The process occurs only at the temperature of the hosts habitat (75–90 °C) and it does not require the presence of the host cell, an exogenous energy source or any cofactors. This host-independent morphological development may be a strategy for viral survival in an environment that is unusually harsh and has limited host availability.

Getting the best out of long-wavelength X-rays: de novo chlorine/sulfur SAD phasing of a structural protein from ATV.

The structure of a 14 kDa structural protein from Acidianus two-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0 A wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structure, one chloride ion turned out to be essential to achieve phasing. The minimal data requirements and the relative contributions of the Cl and S atoms to phasing are discussed. This work supports the feasibility of a systematic approach for the solution of protein crystal structures by SAD based on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization.

Analysis of soil microbial communities based on amplicon sequencing of marker genes

The use of cultivation independent methods has revolutionized soil biology in the last decades. Most popular approaches are based on directly extracted DNA from soil and subsequent analysis of PCR-amplified marker genes by next-generation sequencing. While these high-throughput methods offer novel possibilities over cultivation-based approaches, several key points need to be considered to minimize potential biases during library preparation and downstream bioinformatic analysis. This opinion paper highlights crucial steps that should be considered for accurate analysis and data interpretation.