Sequences and replication of genomes of the archaeal rudiviruses SIRV1 and SIRV2: relationships to the archaeal lipothrixvirus SIFV and some eukaryal viruses

The double-stranded DNA genomes of the viruses SIRV1 and SIRV2, which infect the extremely thermophilic archaeon Sulfolobus and belong to the family Rudiviridae, were sequenced. They are linear, covalently closed at the ends, and 32,312 and 35,502 bp long, respectively, with an A+T content of 75%. The genomes of SIRV1 and SIRV2 carry inverted terminal repeats of 2029 and 1628 bp, respectively, which contain multiple direct repeats. SIRV1 and SIRV2 genomes contain 45 and 54 ORFs, respectively, of which 44 are homologous to one another. Their predicted functions include a DNA polymerase, a Holliday junction resolvase, and a dUTPase. The genomes consist of blocks with well-conserved sequences separated by nonconserved sequences. Recombination, gene duplication, horizontal gene transfer, and substitution of viral genes by homologous host genes have contributed to their evolution. The finding of head-to-head and tail-to-tail linked replicative intermediates suggests that the linear genomes replicate by the same mechanism as the similarly organized linear genomes of the eukaryal poxviruses, African swine fever virus and Chlorella viruses. SIRV1 and SIRV2 both contain motifs that resemble the binding sites for Holliday junction resolvases of eukaryal viruses and may use common mechanisms for resolution of replicative intermediates. The results suggest a common origin of the replication machineries of the archaeal rudiviruses and the above-mentioned eukaryal viruses. About 1/3 of the ORFs of each rudivirus have homologs in the Sulfolobus virus SIFV of the family Lipothrixviridae, indicating that the two viral families form a superfamily. The finding of inverted repeats of at least 0.8 kb at the termini of the linear genome of SIFV supports this inference.     

AFV1, a novel virus infecting hyperthermophilic archaea of the genus acidianus

We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23-130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes.     

Morphology and genome organization of the virus PSV of the hyperthermophilic archaeal genera Pyrobaculum and Thermoproteus: a novel virus family, the Globuloviridae

A novel virus, termed Pyrobaculum spherical virus (PSV), is described that infects anaerobic hyperthermophilic archaea of the genera Pyrobaculum and Thermoproteus. Spherical enveloped virions, about 100 nm in diameter, contain a major multimeric 33-kDa protein and host-derived lipids. A viral envelope encases a superhelical nucleoprotein core containing linear double-stranded DNA. The PSV infection cycle does not cause lysis of host cells. The viral genome was sequenced and contains 28337 bp. The genome is unique for known archaeal viruses in that none of the genes, including that encoding the major structural protein, show any significant sequence matches to genes in public sequence databases. Exceptionally for an archaeal double-stranded DNA virus, almost all the recognizable genes are located on one DNA strand. The ends of the genome consist of 190-bp inverted repeats that contain multiple copies of short direct repeats. The two DNA strands are probably covalently linked at their termini. On the basis of the unusual morphological and genomic properties of this DNA virus, we propose to assign PSV to a new viral family, the Globuloviridae.     

The genome of the archaeal virus SIRV1 has features in common with genomes of eukaryal viruses

The virus SIRV1 of the extremely thermophilic archaeon Sulfolobus has a double-stranded DNA genome similar in architecture to the genomes of eukaryal viruses of the families Poxviridae, Pycodnaviridae, and Asfarviridae: the two strands of the 32,301 bp long linear genome are covalently connected forming a continuous polynucleotide chain and 2029 kb long inverted repeats are present at the termini. Very likely it also shares with these viruses mechanisms of initiation of replication and resolution of replicative intermediates.     

Cleavage of concatemeric DNA at the internal junction of "translocation" mutants of pseudorabies virus and inversion of their L component appear to be linked.

When pseudorabies virus (PrV) strains are grown in chicken embryo fibroblasts (CEF), variants ("translocation" mutants) arise in which there is a duplication of the leftmost sequences of the genome and their translocation in inverted orientation next to the internal inverted repeat bracketing the S component. In these variants, the UL becomes bracketed by inverted repeats and is found in two orientations relative to the Us. To study the cis-functions involved in cleavage of concatemeric DNA as well as those involved in inversion of the L component and to ascertain whether the two events are linked in the "translocation" mutants, a viral mutant (vLD68) was constructed in which the terminal 64 bp of the L component (that include sequences with homology to the pac 2 site of HSV) and the 4 terminal bp of the S component were deleted from the internal junction. Although revertants that have acquired the 68 bp at the internal junction emerge rapidly in populations of vLD68, analysis of the characteristics of this mutant revealed that: (1) the termini derived from both orientations of the L component include the 64 bp that have been deleted from the internal junction of vLD68; (2) in contrast to other "translocation" mutants, the internal junction of the vLD68 genome is not a good substrate for cleavage; (3) inversion of the L component of true vLD68 DNA does not occur or is rare; a good correlation exists in the populations of vLD68 between the proportion of revertants that have acquired an intact internal junction and the proportion of genomes with an L component that inverts. These results show that an intact internal junction in "translocation" mutants is necessary for both inversion of their L components and cleavage at their alternative internal junction. Since cleavage at the alternative junction will result in inversion of the L component, we conclude that inversion of the L component of "translocation" mutants of PrV can be attributed to cleavage of concatemeric DNA at the internal alternative junction.     

Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis

We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.     

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species ofGelasinospora. The plasmid genome is 8231 by long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of theNeurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.     

Sequence and organization of the genomic termini of equine herpesvirus type 1

The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DR1-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the 'a' sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV 'a' sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DR1-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DR1-Uc (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/packaging elements of other herpesviruses are discussed.     

Short inverted repeats function as hotspots of intermolecular recombination giving rise to oligomers of deleted plastid DNAs (ptDNAs)

We have determined the nucleotide sequences around the junction points of oligomeric-deleted ptDNAs possessing a head-to-head or tail-to-tail configuration from long-term cultured cell lines and albino plants. It was shown that DNA rearrangement occurred by direct fusion of deleted ptDNAs in an inverted orientation, which was linked by an asymmetrical sequence of 254–698 bp derived from either of the ptDNAs joined. It is notable that inverted repeats of 7–14 bp flank the asymmetrical sequences at each of the junction points. These features of the DNA sequence around the junction points are commonly observed in oligomeric ptDNA with a large-scale deletion regardless of the cell lines employed. It is suggested that the short inverted repeats are involved in the intermolecular recombination of ptDNA. Received: 1 July / 21 October 1996     

Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability

Alu sequences, the most abundant class of large dispersed DNA repeats in human chromosomes, contribute to human genome dynamics. Recently we reported that long inverted repeats, including human Alus, can be strong initiators of genetic change in yeast. We proposed that the potential for interactions between adjacent, closely related Alus would influence their stability and this would be reflected in their distribution. We have undertaken an extensive computational analysis of all Alus (the database is at http://dir.niehs.nih.gov/ALU) to better understand their distribution and circumstances under which Alu sequences might affect genome stability. Alus separated by <650 bp were categorized according to orientation, length of regions sharing high sequence identity, distance between highly identical regions, and extent of sequence identity. Nearly 50% of all Alu pairs have long alignable regions (>275 bp), corresponding to nearly full-length Alus, regardless of orientation. There are dramatic differences in the distributions and character of Alu pairs with closely spaced, nearly identical regions. For Alu pairs that are directly repetitive, approximately 30% have highly identical regions separated by <20 bp, but only when the alignments correspond to near full-size or half-size Alus. The opposite is found for the distribution of inverted repeats: Alu pairs with aligned regions separated by <20 bp are rare. Furthermore, closely spaced direct and inverted Alus differ in their truncation patterns, suggesting differences in the mechanisms of insertion. At larger distances, the direct and inverted Alu pairs have similar distributions. We propose that sequence identity, orientation, and distance are important factors determining insertion of adjacent Alus, the frequency and spectrum of Alu-associated changes in the genome, and the contribution of Alu pairs to genome instability. Based on results in model systems and the present analysis, closely spaced inverted Alu pairs with long regions of alignment are likely at-risk motifs (ARMs) for genome instability.     

7型腺病毒疫苗株DNA左侧17.5 mu片段克隆及4.8 mu片段核苷酸序列分析

目的获得７型腺病毒疫苗株ＤＮＡ左侧０～１７５ｍｕ片段克隆,分析０～４８ｍｕ片段（末端倒置重复序列、包装信号位点和Ｅｌａ区）核苷酸序列。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ＤＮＡ,将其０３～１７５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果获得了Ａｄ７疫苗株左侧１７５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００、２４０００、２８０００等３个蛋白的ＤＮＡ序列,与Ｇｏｍｅｎ株的同源性分别为９８９％、９７３％和９７５％,推导编码氨基酸的同源性分别为９６６％、９６５％和９６９％。这３个蛋白与Ｇｒｉｄｅｒ株的同源性分别为１００％、９９７％、和９９７％;推导编码氨基酸的同源性分别为１００％、９９１％和９９２％。结论Ａｄ７疫苗株左侧１７３８ｂｐ核苷酸与Ａｄ７Ｇｏｍｅｎ株和Ｇｒｉｄｅｒ株相应片段,具有高度的同源性。     

7型腺病毒疫苗株DNA左侧17.5mu片段克隆及4.8mu片？…

目的 获得７型腺病毒疫苗株ＤＮＡ左侧０－１７．５ｍｕ片段克隆,分析０－４．８ｍｕ片段（末端倒置重复序列,包装信号位点和Ｅｌａ区）核苷酸序列,。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ ＤＮＡ,将其０．３－１７．５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果 获得了Ａｄ７疫苗株左侧１７．５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００,２４０００,２     

Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2

The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.     

The termini of the Chlorella virus PBCV-1 genome are identical 2.2-kbp inverted repeats

The Chlorella virus PBCV-1 genome is a linear nonpermuted 333-kbp dsDNA molecule with covalently closed hairpin termini. The termini (minus the hairpin) are identical inverted repeats of at least 2185 bases after which the sequence diverges. The inverted repeats contain two small potential open reading frames and several direct repeats. However, neither the open reading frames nor the remainder of the inverted repeats are transcribed during PBCV-1 replication. Twenty-nine other Chlorella virus DNAs, of 36 tested, hybridized to the PBCV-1 terminal fragments.     

Identification of the site of recombination in the generation of the genome of DI particles of equine herpesvirus type 1

Defective interfering particles (DIPs) are generated by serial, undiluted propagation of equine herpesvirus type 1 (EHV-1). DIP-rich preparations of EHV-1 mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. The defective genomes consist of reiterations of sequences from the left terminus (0.00 to 0.04 map units) of the long (L) region covalently linked to sequences from the inverted repeats (0.78 to 0.79, 0.83 to 0.87, 0.91 to 0.95, and 0.99 to 1.00 map units) of the short (S) region of the standard genome. We have identified and determined the nucleotide sequences of these segments of the standard genome as well as the component of the defective DNA that contains the site at which these two viral sequences recombined. Comparison of these sequences revealed that there is an 8-nucleotide sequence that is common to both the left terminus sequences and the inverted repeat sequences. These 8-nucleotide identical sequences are located at 3.25 kbp from the left terminus and at 9 kbp downstream of the L-S junction. The recombination between the left terminus and the inverted repeat sequences occurred at the site of homology and resulted in the generation of a novel open reading frame. The last 97 amino acids of an open reading frame of 469 amino acids encoded by sequences within the inverted repeats were replaced by a sequence of 68 amino acids encoded by a 204-bp sequence mapping at 0.023 map units. It will be of interest to determine whether this altered open reading frame, generated by recombination of sequences separated by more than 110,000 bp in the standard genome, plays a role in the varied outcomes of infection mediated by EHV-1 DIPs.     

Sequencing of the bicistronic genome segments S7 and S9 of Mal de Río Cuarto virus (<Emphasis Type="Italic">Fijivirus</Emphasis>, <Emphasis Type="Italic">Reoviridae</Emphasis>) completes the genome of this virus

Summary. The nucleotide sequences of genomic segments S7 and S9 of Mal de Río Cuarto virus (MRCV, Fijivirus group II) have been determined, thus completing the entire genome sequence of the virus. These segments showed a non-overlapping bicistronic structure, as in other members of the genus. MRCV S7 ORF-1 had a length of 1086 bp and encoded a 41.5 kDa putative polypeptide, whereas MRCV S7 ORF-2 had a length of 930 bp and encoded a 36.8 kDa putative polypeptide. Proteins of 39 and 20.5 kDa were predicted for the 1014 bp long MRCV S9 ORF-1 and the 537 bp long MRCV S9 ORF-2, respectively. The terminal 5′ and 3′ sequences of both segments were 5′AAGUUUUU3′ and 5′CAGCUnnnGUC3′, respectively. Specific imperfect inverted repeats of each segment were identified. Comparison of the predicted proteins with those of related virus genome segments counterparts in maize rough dwarf virus (MRDV) and rice black streaked dwarf virus (RBSDV), showed 64.5–44.3% identities. These values are lower than those resulting from comparisons between MRDV and RBSDV. The topology of the trees obtained using the complete nucleotide and amino acid sequences of MRCV S7 and MRCV S9 was consistent with the analysis of the other MRCV segments previously published.     

Molecular analysis of six segments of <Emphasis Type="BoldItalic">Tobacco leaf enation virus</Emphasis>, a novel phytoreovirus from tobacco

Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) associated with TLEV, isolated from infected tobacco in South Africa, suggested that these proteins may correspond to structural Wound tumor virus (WTV) proteins. To further establish the nature of this novel virus disease phenotype in tobacco, molecular characterization of six dsRNA components was undertaken. Full-length cDNA clones were obtained by an optimized modified single-primer amplification sequence-independent dsRNA cloning method. Results of this study revealed the conserved terminal sequence: 5′GG(U/C)...UGAU 3′ of segments S6–S12, while adjacent to these conserved terminal sequences are imperfect inverted repeats (7–15 bp in length), both features being common to reoviruses. The complete nucleotide sequences of segments S5 (2,610 bp), S7 (1,740 bp), S8 (1,439 bp), S10 (1,252 bp), S11 (1,187 bp) and S12 (836 bp) were determined. Comparison of full-length nucleotide sequences with corresponding segments of other phytoreoviruses, Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV) and WTV has shown nucleotide and predicted amino acid identities within the range of 30–60%. TLEV consistently shows a higher identity to WTV than to other phytoreovirus species where sequence data is available. Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of S5 (90.6 kDa); S7 (58.1 kDa); S8 (47.7 kDa); S10 (39.8 kDa); S11 (35 kDa) and S12 (19.5 kDa). The relatively low nucleotide and amino acid identity to other members of the genus demonstrates that TLEV is a novel phytoreovirus, distinct from the only other reported dicotyledenous-infecting WTV and is the first phytoreovirus reported to emerge in Africa.