Download Genetics, biochemistry and structure of the archaeal virus STIV

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Biochemical Society Transactions (2009) Volume 37, part 1
Genetics, biochemistry and structure of the
archaeal virus STIV
Jennifer Fulton*, Brian Bothner†, Martin Lawrence†, John E. Johnson‡, Trevor Douglas†1 and Mark Young*§1
*Department of Microbiology, Montana State University, Bozeman, MT 59715, U.S.A., †Department of Chemistry and Biochemistry, Montana State University,
Bozeman, MT 59715, U.S.A., ‡Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., and §Department of Plant Sciences,
Montana State University, Bozeman, MT 59715, U.S.A.
Abstract
STIV (Sulfolobus turreted icosahedral virus) has been the subject of detailed structural, genetic,
transcriptomic, proteomic and biochemical studies. STIV arguably has been investigated in more detail than
any other archaeal virus. As a result, we know more about STIV than other viruses infecting members of
the Archaea domain. Like most viruses isolated from crenarchaeal hosts, STIV has little in common with
viruses that infect eukaryotic and bacterial hosts and should be considered the founding member of a
new virus family. However, despite this lack of obvious homology with other viruses, STIV has components
of gene content, replication strategy and particle structure reminiscent of viruses of the Eukarya and
Bacteria domains, suggesting an evolutionary relationship between viruses from all domains of life. The
present mini-review describes the current knowledge of this virus and insights it has given us into viral and
cellular evolution, as well as newly developed tools for the further study of STIV–host interactions.
Introduction
STIV (Sulfolobus turreted icosahedral virus) was originally
isolated from an acidic (pH 2.2) high-temperature hot spring
(82◦ C) in Yellowstone National Park [1]. The virus was
isolated from an enrichment culture of an unsequenced
Sulfolobus isolate, YNPRC179. This enrichment culture was
screened by TEM (transmission electron microscopy) for
the presence of virus-like particles which were subsequently
purified and characterized [1].
STIV has a limited host range. The virus has been shown to
infect the Italian isolates Sulfolobus solfataricus strains P1, P2
and a highly susceptible strain derived from S. solfataricus
P2, termed strain P2-2-2-12. Infection of other species,
including Sulfolobus islandicus, Sulfolobus acidocaldarius and
Sulfolobus tokodaii, as well as other uncharacterized Sulfolobus isolates from Yellowstone National Park has been unproductive [1,2]. The full host range of the virus is not known.
STIV is the first lytic virus known to infect a member of
the genus Sulfolobus [3,4]. Infection of S. solfataricus strain
P2-2-2-12 produces plaques and high virus titres in liquid
cultures 36 hpi (hours post-infection). More than 90% of
cells viewed by electron microscopy show evidence of virus
infection (Figure 1). TEM analysis of a time course of the
infection demonstrates lysis of infected cells. Lysis appears
to be an active process directed by virus gene expression,
with alteration of the host S-layer preceding cell membrane
Key words: archaeal virus, archaeon, Sulfolobus solfataricus, Sulfolobus turreted icosahedral
virus (STIV).
Abbreviations used: CRISPR, cluster of regularly interspaced palindromic repeats; dsDNA,
double-stranded DNA; hpi, hours post-infection; MCP, major capsid protein; ORF, open reading
frame; STIV, Sulfolobus turreted icosahedral virus; TEM, transmission electron microscopy.
1
Correspondence may be addressed to either of these authors (email tdouglas@chemistry.
montana.edu or [email protected]).
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Authors Journal compilation disruption. TEM and SEM (scanning electron microscopy)
analyses of these cells show pyramid-like structures on the
cell surface just before lysis. The mechanism by which these
structures form, the host and virus genes that are involved in
the formation of these structures, and the biochemical details
of the lysis process have yet to be elucidated.
STIV genome
The virus encapsidates a circular 17 663 bp dsDNA (doublestranded DNA) genome which replicates episomally with no
evidence of integration into the host genome [1] (Figure 2).
The current annotation of the genome includes 37 ORFs
(open reading frames) [1,5]. Overall, there is little similarity
to ORFs encoded in the STIV genome with other genes
in the databases. However, bioinformatics approaches
have found similarity for three STIV ORFs to proteins
in the public databases (C557, B116 and C92), but each
of these predicted viral proteins is of unknown function
[1]. Another ORF, B164, was predicted by homology
modelling to be an ATPase [5]. The other 33 ORFs have no
homology with proteins in the public databases. The lack
of homology at the genetic and amino acid level to other
known proteins suggests that function must be determined
through further genetic, biochemical and/or structural
analysis.
Virus structure
The virion is ∼74 nm in diameter with an outer protein shell
built on a pseudo T = 31 lattice [6]. This is the first virus
with this symmetry to be described. At each of the 12 5-fold
axes, there are turret-like appendages projecting from the
virion surface from which the virus derives its name. These
Biochem. Soc. Trans. (2009) 37, 114–117; doi:10.1042/BST0370114
Molecular Biology of Archaea
Figure 1 Transmission electron micrographs of S. solfataricus
cells infected with STIV
(A) An infected cell and viruses in various states of assembly at 31 hpi.
(B) A lysed cell and exiting viruses at 48 hpi.
Figure 3 Cryo-electron microscopy reconstruction of STIV
STIV capsid architecture with turrets at each of the 5-fold axes of
symmetry.
Figure 2 STIV genome
Map of the 17 663 bp dsDNA STIV genome. Structural genes (grey
arrows) and non-structural genes (black arrows) are indicated.
5-fold turrets project 13 nm from the virus particle surface
and are approx. 24 nm in diameter at their broadest point
[6]. The function of these turrets is unknown, but one can
speculate that they may be involved in receptor-mediated
cell-surface attachment, injection of DNA into the cell
and/or packaging of DNA into the capsid. MS has identified
11 proteins in the virus particle, nine of which are viral and
two that are host-derived [5]. ORF B345 was identified by
MALDI (matrix-assisted laser-desorption ionization) MS as
the MCP (major capsid protein) of the virion. The MCP is
glycosylated, but the nature of the carbohydrates and the
chemical linkage to the coat protein subunit are unknown [5].
Four ORFs are thought to code for proteins involved with
the turret structure formation, including an ATPase (ORF
B164). The functions of the remaining four virus-coded
proteins are unknown. Two host-derived proteins are found
within the virion. The first is a small DNA-binding protein
(Sso7D), which is likely to be involved with packaging the
viral genome. The second host-derived protein (SSo0881) is a
hypothetical protein with a potential VPS (vacuolar protein
sorting) 24 domain that is found in both Saccharomyces
cerevisiae and mammals and is conserved across the
Sulfolobus family [5]. The virion also contains a host-derived
internal lipid layer surrounding the nucleocapsid genomic
core. This internal membrane comprises a subset of host
lipids that are highly enriched for acidic lipids [6]. The
MCP forming the exterior capsid shell is thought to anchor
to the internal lipid through insertion of its C-terminus into
the lipid layer. The mechanism by which this membrane is
acquired and its function are both unknown, but it is tempting
to speculate that the function of this lipid envelope is to help
protect the viral genome from acid hydrolysis and/or to
help stabilize the virion in its high-temperature environment.
Comparative structural analysis has revealed interesting
insights into virus evolution [5]. The cryo-EM reconstruction
of the virion shows an overall architecture that is similar to
viruses of the other two domains of life (Figure 3). The crystal
structure of the MCP also reflects a common overall structure
that is shared with viruses that infect eukaryotic hosts
(adenovirus) and bacterial hosts (PRD1). This was the first
example of a virus morphology found in all three domains
of life. Structure predictions for several other proteins from
STIV support further this relationship among viruses from all
domains of life. C381 and A223 both share predicted
structural similarity, as determined by fold-recognition algorithms, to the P5 protein of PRD1. The virion architecture,
along with the tertiary structure of the capsid protein itself,
suggests a virus lineage that spans all domains of life, despite
the inability to detect this homology at the sequence level [6].
A number of additional crystal structures exist for
non-structural STIV proteins, which have provided insights
into their function. A197 is a putative glycosyltransferase
[7], and indeed the virus capsid has been demonstrated to
be glycosylated [5]. B116 is a DNA-binding protein [8], and
F93 is a winged-helix DNA-binding protein [9], suggesting
that both may be involved in controlling viral and/or host
gene expression.
Transcriptional analysis
Microarray experiments following an infected culture
and comparing it with an uninfected culture have provided
insights into the timing of events during viral infection as well
as the host’s response to the virus [2]. In a near-synchronous
single replication cycle, there is little evidence for distinct
temporal regulation of viral gene expression. Transcription of
viral genes was first detected 8 hpi, with a peak at 24 hpi.
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Lysis of cells was first detected at 32 hpi. The majority of
the viral genes are co-transcribed between 16 and 24 hpi.
This is in contrast with many DNA viruses of bacteria and
eukarya, which demonstrate distinct temporal patterns of
early and late viral gene expression. A number of putative
operons could be identified based on levels of transcription
and timing. One gene, C67, was transcribed in co-ordination
with the viral structural genes, but has not been identified
as part of the virion. During the infection, 177 host genes
were determined to be differentially expressed, with 124
genes up-regulated and 53 genes down-regulated. The
up-regulated genes were dominated by genes associated with
DNA replication and repair and those of unknown function,
whereas the down-regulated genes, mostly detected at 32 hpi,
were associated with energy production and metabolism. The
observed patterns of transcription suggest that up-regulated
genes are likely to be used by the virus to reprogramme the
cell for virus replication, whereas the down-regulated genes
signal the imminent lysis of the cells. The majority (94%) of
the host genes, however, showed no differential regulation
between infected and uninfected cultures, suggesting that the
virus life cycle is tailored to avoid a host stress response. This
is corroborated further by the absence of typical heat-shockor UV-induction stress-response-type genes. And, in fact,
some genes that are up-regulated under other stresses are
found to be down-regulated in response to STIV infection.
The S. solfataricus P2 genome has six combined CRISPR
(cluster of regularly interspaced palindromic repeats)–Cas
loci which are thought to a form a new viral immunity
system [10]. Several of these CRISPR loci have spacer
sequences that are exact matches to STIV. Despite these
matches, STIV infects S. solfataricus to high levels and no new
STIV-specific spacers were detected post-infection. In fact,
the CRISPR–Cas system was not differentially regulated in
response to STIV infection compared with uninfected cells.
The nature of this system in this host and its interaction
with STIV appears to be modified in comparison with the
systems described in many bacterial species. Unravelling
the function of this system in S. solfataricus should prove to
be an interesting area of research.
STIV genetic system
Further investigation of gene function of the virus depends on
the ability to create site-specific mutations in the viral genome
and analyse the resultant phenotypes. To this end, we have
constructed an infectious clone of the STIV genome. This
clone was constructed by cloning PCR-amplified segments
of the genome into an Invitrogen TA vector. We have demonstrated that the wild-type cloned genome is infectious upon
transformation into host cells. Particles purified from primary transformants can be used as an inoculum for infecting
naı̈ve cells, demonstrating infectivity of the cloned virus.
Strains and species of Sulfolobus that are competent for
transfection with the clone closely follow the host range of
the native virus.
Despite the large insert size for this vector, the construct
appears to be stable and replicates well in Escherichia coli.
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Authors Journal compilation However, the full-length vector does not replicate or produce
particles in S. solfataricus. Removal of the E. coli vector and
re-ligation before electroporation is required for productive
transformation. Additionally, linearized cloned DNA with
or without the E. coli vector does not produce an infection.
This is in contrast with the SSV1 (Sulfolobus spindle-shaped
virus 1)-based shuttle vector that can be transformed as a
linear piece of DNA and transfect the Sulfolobus culture,
albeit at lower efficiency than circular DNA [11].
A number of gene disruption mutant genomes have
been constructed. Initial targets for gene disruptions were
guided by previous studies of the virus. The putative
glycosyltransferase (A197), a DNA-binding protein (B116),
for which structures exist, and C92, which was highly upregulated in the transcriptomics experiments, as well as the
MCP and the putative turret protein (C557), were targeted
for gene disruption by either PCR-based mutagenesis or
digestion and end repair to create frame-shifted mutants. Not
surprisingly, none of the mutants tested to date has produced
viable virus particles or can be detected by qPCR (quantitative
PCR) following transformation into S. solfataricus. However,
a microarray analysis of a time-course transfection with
A197 shows that disruption of the glycosyltransferase
gene still results in viral transcription, indicating that the deficiency in this virus occurs at a downstream point in the
viral replication cycle. We have also constructed a random
library of gene disruptions utilizing a modified Tn5-based
transposon. We are in the process of screening this library
for altered viral phenotypes. Finally, the MCP and C557
are being targeted by PCR-based mutagenesis to look for
regions that might tolerate short peptide insertions.
Conclusions
Recent advances in tool development for both the virus and
host are allowing for a more in-depth understanding of STIV
and its interactions with its host. In particular, the recent
development of genetic tools for both STIV and Sulfolobus
[11–13] should greatly accelerate our understanding of this
unique virus system. The ability to manipulate genetically
the viral genome is an invaluable tool in determining gene
function and elucidating viral life cycle. Detailed genetic
analysis in combination with continued structural, proteomic, transcriptomic and environmental metagenomic studies
will continue to provide insights into the life cycle of STIV.
Despite recent advances, many details of STIV’s replication
cycle, including mechanisms and timing of entry, disassembly,
control of viral transcription, genome replication, assembly and cellular lysis, remain unknown. However, with these
new tools in hand, rapid progress should be forthcoming.
Funding
This work was supported by the National Science Foundation [grant
number EF-0802200] and the Air Force Office of Scientific Research
[grant number FA 9550-07-1-0533].
Molecular Biology of Archaea
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Received 21 August 2008
doi:10.1042/BST0370114
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