Viral Circles Research Articles

Persistent expression of Cotesia plutellae bracovirus genes in parasitized host, Plutella xylostella.

Cotesia plutellae (= vestalis) bracovirus (CpBV) is symbiotic to an endoparasitoid wasp, C. plutellae, and plays crucial roles in parasitism against the diamondback moth, Plutella xylostella. CpBV virion genome consists of 35 circular DNAs encoding 157 putative open reading frames (ORFs). This study re-annotated 157 ORFs with update genome database and analyzed their gene expressions at early and late parasitic stages. Re-annotation has established 15 different viral gene families, to which 83 ORFs are assigned with remaining 74 hypothetical genes. Among 157 ORFs, 147 genes were expressed at early or late parasitic stages, among which 141 genes were expressed in both parasitic stages, indicating persistent nature of gene expression. Relative frequencies of different viral circles present in the ovarian lumen did not explain the expression variation of the viral ORFs. Furthermore, expression level of each viral gene was varied during parasitism along with host development. Highly up-regulated CpBV genes at early parasitic stage included BEN (BANP, E5R and NAC1), ELP (EP1-like protein), IkB (inhibitor kB), P494 (protein 494 kDa) family genes, while those at late stage were mostly hypothetical genes. Along with the viral gene expression, 362 host genes exhibited more than two fold changes in expression levels at early parasitic stage compared to nonparasitized host. At late stage, more number (1,858) of host genes was regulated. These results suggest that persistent expression of most CpBV genes may be necessary to regulate host physiological processes during C. plutellae parasitism.

A Horizontally Transferred Autonomous Helitron Became a Full Polydnavirus Segment in Cotesia vestalis.

Bracoviruses associate symbiotically with thousands of parasitoid wasp species in the family Braconidae, working as virulence gene vectors, and allowing the development of wasp larvae within hosts. These viruses are composed of multiple DNA circles that are packaged into infective particles, and injected together with wasp’s eggs during parasitization. One of the viral segments of Cotesia vestalis bracovirus contains a gene that has been previously described as a helicase of unknown origin. Here, we demonstrate that this gene is a Rep/Helicase from an intact Helitron transposable element that covers the viral segment almost entirely. We also provide evidence that this element underwent at least two horizontal transfers, which appear to have occurred consecutively: first from a Drosophila host ancestor to the genome of the parasitoid wasp C. vestalis and its bracovirus, and then from C. vestalis to a lepidopteran host (Bombyx mori). Our results reinforce the idea of parasitoid wasps as frequent agents of horizontal transfers in eukaryotes. Additionally, this Helitron-bracovirus segment is the first example of a transposable element that effectively became a whole viral circle.

Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases

BackgroundGene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs), symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids’ hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs) has undergone spectacular expansion in several PDV genomes with up to 42 genes.ResultsHere, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication), by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity.ConclusionThe results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in adaptation to host targets.

Replication of phi X174 dna with purified enzymes. I. Conversion of viral DNA to a supercoiled, biologically active duplex.

Conversion of phi X174 viral, single-stranded circular DNA to the duplex replicative form (RF), previously observed with partially purified enzymes, has now been demonstrated with the participation of 12 nearly pure Escherichia coli proteins containing approximately 30 polypeptides. To complete the synthesis of a full length complementary strand, E. coli DNA polymerase I was needed to fill the short gap left by DNA polymerase III holoenzyme, and to remove the primer and replace it with DNA. Production of supercoiled RF required the further actions of E. coli DNA ligase and gyrase. Net synthesis of viral circles was obtained by coupling the formation of RF supercoils to the actions of the phi X174-encoded gene A protein and E. coli rep protein. Viral DNA circles produced from enzymatically synthesized supercoiled RF, serving as template-substrate, were indistinguishable from those produced from RF isolated from infected cells; synthetic RF and the viral circles generated from it by replication were as biologically active in transfection of spheroplasts as the forms obtained from infected cells and virions. The conversion of single-stranded circular DNA to RF is suggested here as a model for discontinuous synthesis of the lagging strand of the E. coli chromosome. The primosome, a complex of some of the replication proteins responsible for initiations of DNA chains, will be described elsewhere. Multiplication of RF supercoils, described in the succeeding paper, proceeds by a rolling-circle mechanism in which the synthesis of viral strands may have analogies to the continuous synthesis of the leading strand of the E. coli chromosome.

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA.

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.

Unique primed start of phage phi X174 DNA replication and mobility of the primosome in a direction opposite chain synthesis.

A specific fragment of the phi X174 viral circle sustains the primed start of complementary DNA strand synthesis in vitro, even though the intact circle permits primed starts at many sites. The 300-nucleotide fragment from restriction nuclease digestion contains the recognition site for protein n', a DNA-dependent ATPase essential for priming phi X174 DNA replication. This n' recognition site contains within it a 44-nucleotide sequence with a potential hairpin structure and may be regarded as the starting signal for replication [Shlomai, J. & Kornberg, A. (1980) Proc. Natl. Acad. Sci. USA 77, 799-803]. After initiation on the 3' side of this sequence, the priming system (primosome) repeatedly generates primers by moving processively on the DNA template in a direction opposite to chain elongation. This primosome mobility is an attractive model for the discontinuous phase of Escherichia coli chromosome replication, in which processive primosome movement with the replicating fork is proposed for repeated initiations of nascent replication fragments.

Initiation of Deoxyribonucleic Acid Synthesis: VII. A UNIQUE LOCATION OF THE GAP IN THE M13 REPLICATIVE DUPLEX SYNTHESIZED IN VITRO

Abstract Replication of M13 viral strands with an enzyme preparation from Escherichia coli produces a duplex (RF–II) with a nearly full length complementary strand (Wickner, W., Brutlag, D., Schekman, R., Kornberg, A., (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 965–969). The gap in this strand relative to the viral circle has a unique location as judged by the following criteria: (a) a Haemophilus influenzae restriction enzyme preparation breaks the RF–II at one specific site near the gap, cleaving the complementary strand into one large and one small fragment; (b) a Haemophilus parainfluenzae restriction enzyme preparation cleaves the RF–I into at least nine fragments, one of which is missing from the RF–II and is restored when the gap is filled by DNA polymerase. These newly added deoxyribonucleotides are primarily associated with the small fragment after cleavage by H. influenzae enzyme, establishing the proximity of this cleavage site near the 3' end of the complementary strand; (c) pyrimidine tracts from the repaired region (and representing the gap) have a simple and distinctive composition, compared with the full complementary strand; and (d) cleavage of the RF–II at the gap by the single-strand-specific endonuclease S1 from Aspergillus oryzae yields linear molecules which after melting and re-annealing from linear rather than circular duplexes, demonstrating that the gap is at only one place relative to the viral strand.

A new form of DNA polymerase 3 and a copolymerase replicate a long, single-stranded primer-template.

A new form of DNA polymerase III, termed Pol III star (Pol III(*)), has been purified to homogeneity from Escherichia coli. Pol III(*) is temperature sensitive when isolated from a thermo-sensitive dnaE mutant, as had been described for Pol III. Pol III(*) and Pol III are separable by gel filtration. Pol III(*) utilizes a duplex template containing short gaps with the same catalytic properties as Pol III. However, Pol III(*) is able to replicate long, singlestranded templates such as homopolymer chains and viral circles of M13 and varphiX174 if provided with the following: spermidine, a primer fragment, and a new protein, termed copolymerase III(*) (Copol III(*)). The latter, purified to homogeneity, has no known independent enzymatic activity and supports synthesis by Pol III(*) but not by Pol I, Pol II, or Pol III.