An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.     

Evolutionary Basis of Codon Usage and Nucleotide Composition Bias in Vertebrate DNA Viruses

Understanding the extent and causes of biases in codon usage and nucleotide composition is essential to the study of viral evolution, particularly the interplay between viruses and host cells or immune responses. To understand the common features and differences among viruses we analyzed the genomic characteristics of a representative collection of all sequenced vertebrate-infecting DNA viruses. This revealed that patterns of codon usage bias are strongly correlated with overall genomic GC content, suggesting that genome-wide mutational pressure, rather than natural selection for specific coding triplets, is the main determinant of codon usage. Further, we observed a striking difference in CpG content between DNA viruses with large and small genomes. While the majority of large genome viruses show the expected frequency of CpG, most small genome viruses had CpG contents far below expected values. The exceptions to this generalization, the large gammaherpesviruses and iridoviruses and the small dependoviruses, have sufficiently different life-cycle characteristics that they may help reveal some of the factors shaping the evolution of CpG usage in viruses. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Application of Motif-Based Tools on Evolutionary Analysis of Multipartite Single-Stranded DNA Viruses

Multipartite viruses contain more than one distinctive genome component, and the origin of multipartite viruses has been suggested to evolve from a non-segmented wild-type virus. To explore whether recombination also plays a role in the evolution of the genomes of multipartite viruses, we developed a systematic approach that employs motif-finding tools to detect conserved motifs from divergent genomic regions and applies statistical approaches to select high-confidence motifs. The information that this approach provides helps us understand the evolution of viruses. In this study, we compared our motif-based strategy with current alignment-based recombination-detecting methods and applied our methods to the analysis of multipartite single-stranded plant DNA viruses, including bipartite begomoviruses, Banana bunchy top virus (BBTV) (consisting of 6 genome components) and Faba bean necrotic yellows virus (FBNYV) (consisting of 8 genome components). Our analysis revealed that recombination occurred between genome components in some begomoviruses, BBTV and FBNYV. Our data also show that several unusual recombination events have contributed to the evolution of BBTV genome components. We believe that similar approaches can be applied to resolve the evolutionary history of other viruses.     

Entry and Disassembly of Large DNA Viruses: Electron Microscopy Leads the Way

Nucleocytoplasmic large DNA viruses are a steadily growing group of viruses that infect a wide range of hosts and are characterized by large particle dimensions and genome sizes. Understanding how they enter into the host cell and deliver their genome in the cytoplasm is therefore particularly intriguing. Here, we review the current knowledge on the entry of two of the best-characterized nucleocytoplasmic large DNA viruses: the poxvirus Vaccinia virus (VACV) and the giant virus Mimivirus. While previous studies on VACV had proposed both direct fusion at the plasma membrane and endocytosis as entry routes, more recent biochemical and morphological data argue for macropinocytosis as well. Notably, direct imaging by electron microscopy (EM) also supported the existence of parallel ways of entry for VACV. Instead, all the giant viruses studied so far only enter cells by phagocytosis as observed by EM, and we discuss the mechanisms for opening of the particle, fusion of the viral and phagosomal membranes and genome delivery via a unique portal, specific for each giant virus. VACV core uncoating, in contrast, remains a morphologically ill-defined process. We argue that correlated light and electron microscopy methods are required to study VACV entry and uncoating in a direct and systematic manner. Such EM studies should also address whether entry of single particles and viral aggregates is different and thus provide an explanation for the different modes of entry described in the literature.     

Behavior of a Virus in a Symbiotic System, Paramecium bursaria—Zoochlorella

SYNOPSIS In a culture system of Paramecium bursaria , virus particles were found in large number. The particle was able to infect and multiply in certain cells of the zoochlorella, an intracellular symbiotic alga of P. bursaria. The infective particle, designated as zoochlorella cell virus (ZCV), was icosahedral and 120–180 nm in edge to edge diameter. The ZCV particle was found to differ from any of the already established viruses attacking the green and the blue-green algae. Within the system where P. bursaria cells were growing, ZCV particles were detected in the depression of the pellicle, among the cilia growing in the cytopharynx, and in the food vacuole of P. bursaria. ZCV particles were infective only for the zoochlorella cells which were recently released from the cytoplasm of P. bursaria. The multiplication process of ZCV comprised the adsorption of the particle to the cell wall of the zoochlorella, the penetration of nucleic acid into the host cell interior, the replication of viral constituents, the maturation of viral particles and their final release by the burst of the zoochlorella cell. ZCV particles appeared only in the cytoplasmic region of the zoochlorella cell in which many ribosomes were distributed. A possible ecosystem among the 3 members consisting of P. bursaria , zoochlorella and ZCV is discussed.     

Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (p = 1.435 g/cm(3)) seems to resemble reovirus cores (p = 1.440 g/cm(3)) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.     

The Extraordinary Evolutionary History of the Reticuloendotheliosis Viruses

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events.     

Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

AT least four established human lymphocyte cell lines, one that originates from a Burkitt's lymphoma and the others from normal persons, contain Epstein-Barr virus (EBV) genome¹. These cells show no viral antigens by immunofluorescence tests nor do they produce virus particles. We are examining one of the four cell lines, Raji (cells from a Burkitt's lymphoma), in more detail. The DNA isolated from purified Raji chromosomes contains as much virus genome as the DNA extracted from whole cells (65 genome equivalents per cell)¹. The viral DNA therefore seems to be in the chromosomes. This result, however, does not necessarily indicate that the viral DNA is physically integrated into chromosomal DNA. The following experiments suggest that the EBV DNA in Raji cells is not covalently linked to the large chromosomal DNA, although the number of viral genomes per cell remains constant during passage. The results do not, however, exclude the possibility that small fragments of cell DNA are bonded to the viral DNA. The data also indicate that EBV DNA in Raji cells exists in strands of complete or nearly complete size.     

Induction of Cellular DNA Synthesis by the Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses

Induction of cellular DNA synthesis by the nondefective adenovirus 2-simian virus 40 hybrid viruses was examined in monkey and hamster cells. It was not possible to discriminate the effects of the hybrid viruses from those of wild-type adenovirus 2.     

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus,Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses. VI. Characterization of the DNA from Five Nondefective Hybrid Viruses

Certain biophysical characteristics of the DNA from each of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND(1), Ad2(+)ND(2), Ad2(+)ND(3), Ad2(+)ND(4), Ad2(+)ND(5)) have been determined. The guanine plus cytosine content varied from 55 to 57% and was not significantly different from that of nonhybrid Ad2 (56%), and the hybrid DNA molecules had mean molecular lengths which were similar to that of the standard, Ad2. The Ad2 and SV40 components of each hybrid were linked by alkali-resistant, presumably covalent bonds. The percentage of SV40 DNA in each hybrid virus was determined by hybridization with SV40 complementary RNA in a calibrated system. The results indicate that each hybrid virus DNA contains a different percentage of SV40 nucleotide sequences. The estimated size of the SV40 DNA component varies from 48,000 daltons for Ad2(+)ND(3) to 840,000 daltons for Ad2(+)ND(4), the latter being equivalent to between one-fourth and one-third of the SV40 genome.     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.     

Lack of Sequence Homology Between the 70S RNA of Various RNA Tumor Viruses and the DNA of Simian Virus 40 or Polyoma Virus

No significant hybridization was detected of DNA from simian virus 40 or polyoma virus, and of 70S RNA from avian myeloblastosis virus, murine leukemia virus (Rauscher), murine sarcoma virus (Kirsten), RD-114B, simian sarcoma virus-1, or Mason-Pfizer virus.     

Correlation Between Epstein-Barr Virus Membrane Antigen and Three Large Cell Surface Glycoproteins

A correlation between Epstein-Barr virus membrane antigen (MA) and three surface glycoproteins has been established on the basis of radio-immunoprecipitation and immunoabsorption experiments. For radio-immunoprecipitation, Epstein-Barr virus-infected cells were radiolabeled either with neuraminidase-galactose oxidase tritiated borohydride, a procedure highly specific for surface glycoproteins, or with a general tritiated amino acid mixture. Intact cells were incubated with MA(-) or MA(+) human sera, washed free of unbound immunoglobulins, and then lysed with Nonidet P-40. The antigen-antibody complexes were bound to protein A-Sepharose and after elution with sodium dodecyl sulfate were analyzed by acrylamide gel electrophoresis in sodium dodecyl sulfate. MA(+) sera specifically precipitated three glycoproteins with molecular weights of 236,000, 212,000, and 141,000 from B95-8 cells induced with 12-O-tetradecanoylphorbal-13-acetate (TPA) and from Raji cells superinfected with P3HR-1 virus. These glycoproteins were not detected on Epstein-Barr virus-negative Ramos cells treated with TPA or on B95-8 cells treated simultaneously with TPA and phosphonoacetic acid. Soybean lectin-Sepharose bound all three glycoproteins, and lectin-Sepharose-bound glycoproteins from TPA-induced P95-8 cells absorbed MA-specific antibody from MA(+) human sera. The data strongly suggest that either all three glycoproteins have MA determinants or they are part of a complex in which one or more of the components constitute the reactive antigen.