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At least 19 recordsLinked to original sources
PubMed2002

Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane.

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.

Bacteriophage PRD1↗
PubMed2005

Response of four types of coliphages to high hydrostatic pressure.

Pressure inactivation of four types of coliphages, varphiX 174 (ssDNA virus), MS2 (ssRNA virus), lambda imm434 (dsDNA virus) and T4 (dsDNA virus), was studied to evaluate their potential as human enteric viral surrogates for use in validation of commercial pressure processing treatments. Phage varphiX 174 demonstrated an unexpected high resistance to pressure with no more than 1-log(10) reduction observed following exposures to 350-600 MPa. There was no greater than 1-log(10) reduction below 500 MPa for MS2 in modified phosphate-buffered saline, but a 3.3-log(10) reduction was observed for MS2 pressurized at 600 MPa. Coliphages lambda imm434 and T4 were relatively sensitive to pressure in demonstrating inactivation at 350 MPa. At 21 degrees C, lambda imm434 was inactivated in modified phosphate-buffered saline or Dulbecco's Modified Eagle's Medium plus 5% fetal bovine sera by at least 7.5-log(10) when exposed to 400 MPa for 5 min. Treatment at 450 MPa for 5 min was necessary to obtain a log(10) reduction of 6-7 for T4.

Analysis of Variance↗
PubMed2005

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange.

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.

Amino Acid Sequence↗
PubMed2006

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry.

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.

Amino Acid Substitution↗
PubMed2006

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life.

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

Amino Acid Sequence↗
PubMed2006

Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1.

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146 bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

Animals↗
PubMed2003

Only one pRNA hexamer but multiple copies of the DNA-packaging protein gp16 are needed for the motor to package bacterial virus phi29 genomic DNA.

A common feature in the maturation of linear dsDNA viruses is that the lengthy viral genome is translocated with remarkable velocity into a limited space within a preformed protein shell using ATP as motor energy. Most biomotors, such as myosin, kinesin, DNA-helicase, and RNA polymerase, contain one ATP-binding component that acts processively. An examination of the well-studied dsDNA viruses reveals that DNA packaging motors involve two nonstructural components. Which component of the motor is the integrated processive factor to turn the motor has not been identified. In bacterial virus phi 29, these two components consist of a gp16 protein and an RNA molecule called pRNA. We have previously predicted and recently confirmed that gp16 binds ATP. It is generally believed that gp16 serves as an ATP-binding and processive component to drive the motor. In this article, phi 29 DNA-packaging intermediates were purified in quantity and examined to differentiate the role between gp16 and pRNA. It was found that the pRNA hexamer is an integral motor component, while gp16 is not stably bound. Only one pRNA hexamer, but multiple copies of gp16, were needed to accomplish DNA packaging. pRNA functions continuously during the entire DNA translocation process, suggesting that pRNA is a vital part of the DNA packaging motor.

Bacillus Phages↗
PubMed2006

Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system.

The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.

Biological Assay↗
PubMed2005

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

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.

Amino Acid Sequence↗
PubMed1996

Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes.

In order to examine genetic relatedness among viruses that infect microalgae, DNA polymerase gene (DNA pol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (Chlorella strains NC64A and Pbi, Micromonas pusilla and Chrysochromulina spp.). Phylogenetic trees based on DNA pol sequences and hybridization of total genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequence data showed good concordance (r=0.90), indicating that DNA pol sequences can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses. The phylogenetic tree inferred from the deduced amino acid sequences of DNA pol from 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses, and African swine fever virus corresponded well with groupings based on the International Committee on Taxonomy of Viruses. Microalgal viruses are more closely related to each other than to the other dsDNA viruses and form a distinct phyletic group, suggesting that they share a common ancestor and belong to the Phycodnaviridae. Moreover, the Phycodnaviridae are more closely related to the Herpesviridae than to other virus families for which DNA pol sequences are available.

Amino Acid Sequence↗
PubMed2006

RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins.

RNA silencing is an ancient mechanism of gene regulation with important antiviral roles in plants and insects. Although induction of RNA silencing by RNA viruses has been well documented in plants, the interactions between DNA viruses and the host silencing machinery remain poorly understood. We investigate this question with cauliflower mosaic virus (CaMV), a dsDNA virus that expresses its genome through the polycistronic 35S RNA, which carries an unusually extensive secondary structure known as translational leader. We show that CaMV-derived siRNAs accumulate in turnip- and Arabidopsis-infected plants and that the leader is a major, albeit not exclusive, source for those molecules. Biogenesis of leader-derived siRNA requires the coordinated and hierarchical action of the four Arabidopsis Dicer-like (DCL) proteins. Our study also uncovers a "facilitating" role exerted by the microRNA biosynthetic enzyme DCL1 on accumulation of DCL2-, DCL3-, and DCL4-dependent siRNAs derived from the 35S leader. This feature of DCL1 defines a small RNA biosynthetic pathway that might have relevance for endogenous gene regulation. Several leader-derived siRNAs were found to bear near-perfect sequence complementarity to Arabidopsis transcripts, and, using a sensor transgene, we provide direct evidence that at least one of those molecules acts as a bona fide siRNA in infected turnip. Extensive bioinformatics searches identified >100 transcripts potentially targeted by CaMV-derived siRNAs, several of which are effectively down-regulated during infection. The implications of virus-directed silencing of host gene expression are discussed.

Arabidopsis↗
PubMed1991

Antibodies to viral and mammalian native DNA in response to BK virus inoculation and subsequent immunization with calf thymus DNA.

Recent studies have demonstrated that anti-DNA antibodies share important genetical features with antibodies to exogenous antigens, suggesting that anti-DNA antibody responses may be (auto-) antigen driven. We have earlier defined three out of five rabbits as anti-dsDNA antibody responders based on reactivity with calf thymus (CT) dsDNA after inoculation with the human dsDNA virus BK. In the present study we demonstrate that all five animals that received BK virus inoculations produced antibodies to BK virus dsDNA. These antibodies did not cross-react with CT dsDNA, as shown by inhibition experiments. The anti-BK dsDNA antibodies persisted over time, in contrast to the anti-CT dsDNA antibodies that decreased shortly after a peak following the first boost of BK virus. While the anti-CT dsDNA antibodies decreased, the anti-BK dsDNA antibodies remained elevated, thus supporting the results of the inhibition experiments which showed that two independent antibody populations are produced after BK virus inoculations. In the three animals producing anti-mammalian dsDNA antibodies, antibodies recognizing CT dsDNA reappeared after intravenous administration of a complex of CT dsDNA and methylated bovine serum albumin (MBSA) without adjuvant. The latter anti-CT dsDNA antibodies did not cross-react with BK dsDNA. In contrast to earlier studies we conclude that mammalian dsDNA may be immunogenic, and that discrete molecular differences in DNA antigens from different sources may induce anti-dsDNA antibodies specific for dsDNA molecules of different origin.

Animals↗
PubMed2005

Gene-expression profiling of White spot syndrome virus in vivo.

White spot syndrome virus, type species of the genus Whispovirus in the family Nimaviridae, is a large, double-stranded DNA (dsDNA) virus that infects crustaceans. The genome of the completely sequenced isolate WSSV-TH encodes 184 putative open reading frames (ORFs), the functions of which are largely unknown. To study the transcription of these ORFs, a DNA microarray was constructed, containing probes corresponding to nearly all putative WSSV-TH ORFs. Transcripts of 79 % of these ORFs could be detected in the gills of WSSV-infected shrimp (Penaeus monodon). Clustering of the transcription profiles of the individual genes during infection showed two major classes of genes: the first class reached maximal expression at 20 h post-infection (p.i.) (putative early) and the other class at 2 days p.i. (putative late). Nearly all major and minor structural virion-protein genes clustered in the latter group. These data provide evidence that, similar to other large, dsDNA viruses, the WSSV genes at large are expressed in a coordinated and cascaded fashion. Furthermore, the transcriptomes of the WSSV isolates WSSV-TH and TH-96-II, which have differential virulence, were compared at 2 days p.i. The TH-96-II genome encodes 10 ORFs that are not present in WSSV-TH, of which at least seven were expressed in P. monodon as well as in crayfish (Astacus leptodactylus), suggesting a functional but not essential role for these genes during infection. Expression levels of most other ORFs shared by both isolates were similar. Evaluation of transcription profiles by using a genome-wide approach provides a better understanding of WSSV transcription regulation and a new tool to study WSSV gene function.

Animals↗
PubMed2003

Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria.

Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.

Amino Acid Sequence↗
PubMed2002

Coccolithovirus (Phycodnaviridae): characterisation of a new large dsDNA algal virus that infects Emiliana huxleyi.

Emiliania huxleyi-specific viruses ( EhV) were isolated from E. huxleyi blooms off the coast of Plymouth, UK, in July 1999 and July/August 2001, and from an E. huxleyi bloom induced during a mesocosm experiment in a fjord off Bergen, Norway, during June 2000. Transmission electron microscopy revealed that all 10 virus isolates are 170-200 nm in diameter with an icosahedral symmetry. Their density is approximately 1.2 in CsCl gradients and they have large double stranded DNA genomes approximately 410 kb in size. Phylogenetic analysis of the DNA polymerase genes of these viruses suggests that EhV belongs to a new genus within the family of algal viruses, Phycodnaviridae. We propose to name this new virus genus Coccolithovirus. Differences within members of the Coccolithovirus were elucidated by host range analysis of the virus isolates and sequence analysis of a gene fragment encoding part of their putative major capsid protein. All 10 virus isolates within this new genus only infected E. huxleyi strains that have previously been shown to exhibit low dimethylsulphoniopropionate lyase (DMSP-lyase) activity (CCMP1516, CCMP374 and L), while E. huxleyi strains with high DMSP-lyase activity (CCMP373 and CCMP379) were resistant to infection.

Base Sequence↗
PubMed2003

Use of PEG to acquire highly soluble DNA-packaging enzyme gp16 of bacterial virus phi29 for stoichiometry quantification.

All linear dsDNA viruses package their genome into a preformed procapsid via a ATP-driving motor involving two nonstructural enzymes or ATPase. This essential viral replication step has been investigated in the quest for new antiviral drugs. These DNA-packaging motors could be potential parts in nanotechnology. But both the low solubility and self-aggregation of all nonstructural enzymes have seriously hampered studies on these motors. Bacterial virus phi29 DNA-packaging motor has been well characterized. But the role of the nonstructural ATPase gp16 has not been well defined due to its hydrophobicity, low solubility, and self-aggregation. Here we report a novel approach to obtain affinity-purified, soluble, and highly active native gp16 with the aid of polyethylene glycol (PEG) or acetone. With several thousand-fold increase in specific activity in comparison to the traditional method, this unique approach has made the quantification of gp16 feasible. The basic functional unit of gp16 in solution was found to be a monomer, as determined by sedimentation and size exclusion chromatography. This result leads to a subsequent finding that the stoichiometry of gp16 for phi29 DNA-packaging was about 11+/-2. These findings will facilitate the study on this novel motor that involves three pRNA dimers and a 12-subunit connector.

Adenosine Triphosphatases↗
PubMed2001

Chemical modification patterns of active and inactive as well as procapsid-bound and unbound DNA-packaging RNAof bacterial virus Phi29.

During replication, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into the limited space within the preformed procapsid. We previously found that a viral-encoded RNA (pRNA) played a key role in bacterial virus phi29 DNA translocation. Design of mutant pRNA sets containing two and three inactive mutant pRNAs, respectively, led to the conclusion that the stoichiometry of pRNA in DNA packaging is the common multiple of 2 and 3. Together with studies using binomial distribution of mutant and wild-type pRNA, it has been confirmed that six pRNAs of phi29 form a hexagonal complex to drive the DNA translocating machine. These findings have brought about commonality between viral DNA packaging and other universal DNA/RNA-riding processes including DNA replication and RNA transcription. Chemical modification was used to compare the structures of active and inactive as well as free and procapsid-bound pRNA. Our results explain why certain pRNA mutants are inactive in DNA packaging while remaining competent in procapsid binding, since the mutations were located in a domain involved in DNA translocation that is dispensable for procapsid binding. A mutant pRNA that had reduced procapsid binding was revealed to have a structural alteration within the procapsid-binding region that may account for the binding deficiency. Chemical probing of procapsid-bound pRNA revealed a large area of protection, while a 3-base bulge, C(18)C(19)A(20), was accessible to chemicals. A pRNA with a deletion of this 3-base bulge was fully competent to form dimers, bind procapsids, and inhibit phi29 virion assembly in vitro; however, its activity in DNA packaging and virion assembly was completely lost. The results suggest that this bulge is not involved in procapsid binding but may interact with other DNA-packaging components. A computer model showing the location of the CCA bulge was presented.

Bacillus Phages↗