Mechanisms of spiroplasma genome variation associated with SpV1-like viral DNA inferred from sequence comparisons.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral sequences in genome evolution, we examined available nucleotide sequences of viruslike elements in the S. citri chromosome. Comparison of integrated and nonintegrated sequences of spiroplasma virus SpV1-C74 DNA suggested that it is an encapsidated form of the circular transposition intermediate belonging to an insertion sequence (IS3) family member. One SpV1-C74 viral DNA fragment was identified as interrupting the remains of a DNA adenine modification methylase gene. A viral DNA insertion of SpV1-R8A2 B DNA had hallmarks of having suffered an internal deletion by a site-specific recombination system. Homologous recombination likely was responsible for several deletions within viral DNA. A homologous recombination event was inferred between part of a viral DNA insertion and a similar chromosomal sequence. Dispersed sequences from SpV1-like C4 open reading frames (ORFs) were identified as involved in a complex deletion-inversion event. Thus, SpV1-like sequences likely have altered spiroplasma genomes by inserting within active genes, destroying their function, by providing targets for site-specific recombination, by mediating deletions of sequences adjacent to their integration sites, and by providing targets for homologous recombination, leading to inversions.     

Comparative genomics of foot-and-mouth disease virus

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5' and 3' untranslated regions (UTR), as was tolerance for insertions/deletions in the 5' UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L(pro), 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.     

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.     

Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles

The genomes of defective-interfering (DI) particles derived from the Sabin strain of type 1 poliovirus (PV1(Sab] were characterized by nuclease S1 mapping using complementary DNA (cDNA) copies of PV1(Sab) genome as probes. The results demonstrated variety in the size and location of the deletions, which were compatible with our previous prediction. The results further indicated that the locations of the deletions were limited within the internal genome region encoding viral capsid proteins and that the deletion sites were clustered in certain areas on the genome. Sequence analysis of a number of cloned cDNAs to the DI genomes revealed that every DI genome retained the correct reading frame for viral protein synthesis. These results strongly suggested that one or all of the viral non-structural proteins might be cis-acting at least at a certain stage in viral replication. A computer search for secondary structures with regard to the deletion sites provided a possible common structure from which, supported by sequences existing on the plus or minus RNA strand of PV1(Sab), deletion regions looped out from the remaining sequences. Replicase might, therefore, skip these transiently formed loop structures with certain frequencies, resulting in the generation of DI genomes. This model could also be considered as a model for genetic recombination in these RNA genomes. Possible "supporting sequences" were also found for every rearranged site on the RNAs of influenza virus and sindbis virus. Thus, we propose a new copy-choice model, designated the "supporting sequence-loop model", for the generation of rearrangements occurring on single-stranded RNA genomes.     

Nonhomologous RNA recombination during negative-strand synthesis of flock house virus RNA.

During sequential replicative passages of viral RNA from the nodavirus flock house virus, spontaneous deletion of RNA sequences occurred frequently. Families of deleted RNA molecules were derived from both segments of the bipartite viral genome and found to contain single, double, or triple deletions. These deletions were attributed to template switching by the flock house virus RNA replicase, resulting in recombination between distant sequences and excision of the intervening nucleotides. From sequence analysis of the recombination junctions, we concluded that the process of template switching was influenced by both the primary sequence and the secondary structure of the RNA and that it occurred predominantly during synthesis of RNA negative strands.     

Coordinated Function of Cellular DEAD-Box Helicases in Suppression of Viral RNA Recombination and Maintenance of Viral Genome Integrity

The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5’ end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication.     

Synchronous loss of quasispecies memory in parallel viral lineages: a deterministic feature of viral quasispecies

Viral quasispecies are endowed with a memory of their past evolutionary history in the form of minority genomes of their mutant spectra. To determine the fate of memory genomes in evolving viral quasispecies, we have measured memory levels of antigenic variant of foot-and-mouth disease virus (FMDV) RED, which includes an Arg-Glu-Asp (RED) at a surface antigenic loop of the viral capsid. The RED reverted to the standard Arg-Gly-Asp (RGD), and the RED remained as memory in the evolving quasispecies. In four parallel evolutionary lineages, memory reduction followed a strikingly similar pattern, and at passage 60 memory levels were indistinguishable from those of control populations (devoid of memory). Nucleotide sequence analyses indicated that memory loss occurred synchronously despite its ultimate molecular basis being the stochastic occurrence of mutations in the evolving quasispecies. These results on the kinetics of memory levels have unveiled a deterministic feature of viral quasispecies. Molecular mechanisms that may underlie synchronous memory loss are the averaging of noise signals derived from mutational input, and constraints to genome diversification imposed by a nucleotide sequence context in the viral genome. Possible implications of the behaviour of complex, adaptive viral systems as experimental models to address primary mechanisms of neurological memory are discussed.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Homologous recombination between different genotypes of hepatitis B virus

Phylogenetic analysis was used to examine the evolutionary relationships among 99 complete HBV sequences. Analysis revealed nine viral genomes clustered with different genotypes depending on genome region analyzed. This discordance indicated that recombination events occurred during HBV history. The putative breakpoints between genomes of different genotypes have been mapped. Six mosaic genomes representing B/C hybrids were isolated in East Asia and three A/D hybrids in Italy. At least some recombinant strains appear to be fully viable and possess high evolutionary potential. As a result, B/C recombinants overspread through the East Asia region. They were found among the isolates from Japan, China and Indonesia. Our results suggest that recombination is a significant and relatively frequent event in the evolution of HBV genome. A possible mechanism and the implications of recombination for the natural history of HBV, clinically important properties, and phylogenetic reconstruction are discussed.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Coenzymes, viruses and the RNA world

The results of a detailed bioinformatic search for ribonucleotidyl coenzyme biosynthetic sequences in DNA- and RNA viral genomes are presented. No RNA viral genome sequence available as of April 2011 appears to encode for sequences involved in coenzyme biosynthesis. In both single- and double-stranded DNA viruses a diverse array of coenzyme biosynthetic genes has been identified, but none of the viral genomes examined here encodes for a complete pathway. Although our conclusions may be constrained by the unexplored diversity of viral genomes and the biases in the construction of viral genome databases, our results do not support the possibility that RNA viruses are direct holdovers from an ancient RNA/protein world. Extrapolation of our results to evolutionary epochs prior to the emergence of DNA genomes suggest that during those early stages living entities may have depended on discontinuous genetic systems consisting of multiple small-size RNA sequences.     

The nucleotide sequence of repetitive monkey DNA found in defective simian virus 40.

DNA fragments containing monkey DNA sequences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was determined using both RNA and DNA sequencing methods. The nucleotide sequence of this homogeneous DNA segment does not contain discernible multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration frequency of the sequence in the monkey genome is >10⁶. Digestion of total monkey DNA (from uninfected cells) with endonuclease R Hind III produces relatively large amounts of discrete DNA fragments that contain extensive regions homologous to the fragment isolated from the defective SV40 DNA.A second fragment, also containing monkey sequences, was isolated from the same defective substituted SV40 genome. The nucleotide sequence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined.The sequences in both fragments are also present in other, independently derived, defective substituted SV40 genomes.     

Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVPS/1/P2 (EcoRI res).

The entire nucleotide sequence (1210-base-pair repeating units) of a defective variant of simian virus 40 is presented. Within this variant there are deletions of large portions of the wild type genome and an inversion within the remaining wild type viral sequences. In addition, the defective variant contains DNA sequences derived from the permissive monkey cells in which the virus was propagated. The monkey sequences include a portion that is homologous to sequences within highly repeated monkey DNA (alpha component) as well as portions derived from sequences that are infrequently repeated in the monkey genome. One out of every three to four of the tandem 1210-base-pair repeat units contains in addition, a duplication of a part of the monkey sequences. The sequence information defines the structures of a number of recombinational joints which result from deletions, inversions, duplications, and insertions of host sequences into the viral genome. The data demonstrate that the various recombinational events that resulted in the formation of this defective variant did not depend on extensive homology between recombining segments.     

Normalization of Complete Genome Characteristics: Application to Evolution from Primitive Organisms to Homo sapiens

Normalized nucleotide and amino acid contents of complete genome sequences can be visualized as radar charts. The shapes of these charts depict the characteristics of an organism’s genome. The normalized values calculated from the genome sequence theoretically exclude experimental errors. Further, because normalization is independent of both target size and kind, this procedure is applicable not only to single genes but also to whole genomes, which consist of a huge number of different genes. In this review, we discuss the applications of the normalization of the nucleotide and predicted amino acid contents of complete genomes to the investigation of genome structure and to evolutionary research from primitive organisms to Homo sapiens. Some of the results could never have been obtained from the analysis of individual nucleotide or amino acid sequences but were revealed only after the normalization of nucleotide and amino acid contents was applied to genome research. The discovery that genome structure was homogeneous was obtained only after normalization methods were applied to the nucleotide or predicted amino acid contents of genome sequences. Normalization procedures are also applicable to evolutionary research. Thus, normalization of the contents of whole genomes is a useful procedure that can help to characterize organisms.     

The mechanism of transduction of proto-oncogene c-src by avian retroviruses

Chicken c-src sequences have been transduced by avian leukosis viruses (ALV) and by partial src-deletion (td) mutants of Rous sarcoma virus in several independent events. Analyses of the recombination junctions in the genomes of src-containing viruses and the c-src DNA have shed light on the mechanism of transduction, which involves at least two steps of recombination. The initial recombination between a viral genome and the 5' region of c-src appears to occur at the DNA level. This step does not require extensive homology and can be mediated by stretches of sequences with only partial homology. The 5' recombination junction can also be formed by splicing between viral and c-src sequences. The second recombination is presumed to occur between the transducing ALV or td viral RNA and the viral-c-src hybrid RNA molecule generated from the initial recombination. This step involving recombination at the 3' ends of those molecules restores the 3' viral sequences essential for replication to the viral-c-src hybrid molecule. High frequency of c-src transduction by partial td mutants suggests that the second recombination is greatly enhanced when there is sequence homology between the transducing virus and the 3' region of c-src. Incorporation of the c-src sequences into an ALV genome results in greatly elevated expression of the gene. However, increased expression of c-src alone is insufficient to activate its transforming potential. Structural changes in c-src are necessary to convert it into a transforming gene. The changes can be as small as single nucleotide changes resulting in single amino aid substitutions at certain positions. Mutations can occur rapidly during viral replication after c-src is incorporated into the viral genome. Therefore, it is most likely that transduction of c-src by ALV is followed by subsequent mutation and selection for the sarcomagenic virus. In the case of transduction by td viruses that retain certain src sequences, joining of these sequences with the transduced c-src apparently is sufficient to activate its transforming potential.