Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Integrative recombination between adenovirus type 12 DNA and mammalian DNA in a cell-free system: joining by short sequence homologies

A cell-free system was developed to investigate the mechanism of how junctions are formed between viral and cellular DNAs during adenoviral DNA integration into the hamster cell genome. Recombination between the segment of adenovirus type 12 (Ad12) DNA, that comprises sequence coordinates 20 885–24 053, subsequently termed PstI-D fragment and the hamster preinsertion DNA sequence p7 was studied in a cell-free system. The p7 DNA segment had served as viral DNA integration site in the Ad12-induced tumor CLAC1. Nuclear extracts initially from uninfected BHK21 hamster cells were fractionated by a series of chromatographic steps. DNAs of the in vitro generated recombinants were analyzed in detail. In the course of the recombination reaction, the two linear molecules were joined. The reaction took place between two short homologous sequences one of which was always at or very close to a DNA terminus, the second one could be several kilobase pairs remote from a DNA terminus. Apparently, the nucleotide sequence at the terminus of one recombining molecule determined the point of junction by searching for short homologies in the partner molecule. The recombination reaction was not conservative, the sequences in-between the short sequence homologies and one of the short sequence homologies were deleted in the in vitro recombinants. Two main criteria influenced the choice of interacting short sequence homologies: perfect homologies of 8–9 bp were most frequently found, they were preferred over more extended, but less perfect homologies. Comparing different short sequence homologies with similar stabilities, those combinations seemed to be chosen in the reaction which led to a minimal loss of nucleotides in the recombinants. The in vitro activity was found in nuclear extracts from both hamster and human cells. The activity was, hence, available for Ad12 DNA in productively infected human and abortively infected hamster cells. The specific recombination activity was increased in nuclear extracts of hamster cells abortively infected with Ad12. The junction sites in the recombinants, which were generated by the cell-free system, were very similar to junctions between adenoviral and cellular DNAs cloned from Ad12-induced tumor cells and Ad12-transformed cell lines.     

Polarity in adenovirus recombination

The distributions of the crossovers necessary to generate ts+ genomes have been examined in a collection of clonally unrelated ts+ recombinants from a set of ts X ts adenovirus crosses. In a cross between two parents that are grossly heterologous between map units 80.2 and 91.5, the distribution of crossovers was significantly skewed toward the left-hand end of the genome, with a declining frequency proceeding rightward. This gradient of recombination was modified by the removal of the right-hand heterology and by the presence of another region of heterology between map units 3.67 and 10.11. In a cross where the ts markers were flanked by both heterologies, no gradient was observed and ts+ recombinants were characterized by a higher rate of supernumerary crossovers. In a cross designed so that one ts marker was internal to two heterologies, crossovers were found disproportionately between the second ts marker and the nearby heterology. In addition, ts+ recombinants formed by crossing over internal to the heterologies again were accompanied by a high frequency of supernumerary crossovers. Finally, ts+ recombinant frequencies in crosses identical except for the presence of either one or two flanking heterologies were markedly lower in the latter case. These data, taken together, suggest that a major pathway of adenovirus recombination initiates at, or near, the molecular termini and is perhaps driven by the displaced single strands produced during DNA replication. Internal initiation, on the other hand, may employ these single strands to form genetic "patches."     

Location and nucleotide sequence of two tRNA genes and a tRNA pseudo-gene in the maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria

We report the nucleotide sequence of three tRNA genes from maize mitochondria. The genes are located in two BamHI fragments, 3.55 and 5.7 kb long, adjacent to the S2 sequence in the maize mitochondrial genome. On the 3.55 kb BamHI fragment, we have characterized a tRNA(Cys)(GCA) gene. A strong sequence homology of this tRNA(Cys)(GCA) gene with its chloroplast counterpart in wheat suggests that it may be part of a chloroplast DNA insertion into the mitochondrial genome. This gene has been found to be transcribed in the mitochondrion. Two tRNA genes are located on the 5.7 kb BamHI fragment, separated from each other by 250 bp. One is a mitochondrial tRNA(Ser)(GCU) gene. The other, a non-transcribed tRNA(Phe)-like gene, is interrupted by a 49 base-pair inserted DNA sequence in the variable loop and has a Leu (UAA) anticodon.     

Identification and nucleotide sequence of the early region 1 from canine adenovirus types 1 and 2

The genome of canine adenovirus type 1 (CAV-1) has been cloned and restriction maps compiled. These maps are compared with those of canine adenovirus type 2 (CAV-2). The left ends of both genomes were further characterised by DNA sequence analysis. Several features of the DNA sequence and predicted polypeptide sequence are similar to those of the human adenoviruses. The level of homology observed across the E1 regions appears to be of the same order as the overall DNA similarity between CAV-1 and CAV-2 (75%). Transfection experiments using the presumptive E1a containing region of CAV-2 suggests that it encodes a transactivating function typical of the human adenovirus E1a genes.     

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.     

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.     

Prevalence and characterization of enteric adenoviruses in the South of Ireland

Enteric adenoviruses have been shown to be a substantial cause of pediatric gastroenteritis in various parts of the world, and are considered to be the second most common cause of viral gastroenteritis, next to rotavirus in young children. Genetic characterization of 95 adenovirus isolates obtained from patients with acute gastroenteritis between 2002 and 2007 from the southern regions of Ireland, were characterized by PCR analysis, restriction endonuclease (RE) analysis and sequencing analysis. All isolates were found to be of adenovirus type 41 origin. Genetic analysis of seven hypervariable regions (HVRs) located within the hexon gene has revealed a high level of amino acid sequence homology in samples over the course of this study, with a very close relationship to the D22 genome type. The D22 genome type has been detected in several other countries, thus suggesting Irish isolates have common genome types with other stains worldwide. This is the first such study undertaken in the south of Ireland, to type and genetically characterize adenoviral gastroenteritis isolates, and has revealed a high level of conservation within the isolated analyzed.     

Conserved sequences of the adenovirus genome for detection of all human adenovirus types by hybridization.

The application of DNA hybridization directly to clinical specimens has the potential of improving the diagnosis of fastidious types of adenovirus. In this study, the genome of one adenovirus type from each human subgenus (A to F) was systematically evaluated by hybridization for homologous sequences to find the optimal common probe for detection of all human adenovirus types. The area of cross-hybridization, most closely defined with adenovirus type 2 (Ad2), mapped from map units 11.4 to 16.1 and 26.9 to 29.7 and, principally, to a central area of the genome between map units 47.5 and 65.2. The last area, enclosing the hexon gene, was highly conserved. Cloned probes generated from this area demonstrated the greatest homology to heterologous types by hybridization analysis. A HindIII-BglII clone containing the hexon gene of Ad2 within narrow confines reacted most evenly with all adenoviral types and detected the DNA of all other subgenera with a sensitivity 2 logs greater than that of a complete genomic Ad2 probe. The most homologous adenoviral gene sequences were observed in genes involved with DNA replication or intimately connected to the hexon in the early capsid formation. These results show that the hexon gene constitutes the best single region of the adenovirus genome for use as a genus-specific probe for the diagnosis of all human adenoviral subgenera by DNA hybridization.     

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.     

Recombination in adenovirus: crossover sites in intertypic recombinants are located in regions of homology

Wild-type recombinants have been selected from intertypic genetic crosses between Ad2 and Ad5 ts mutants, with lesions within or close to the hexon gene, and the structure of the recombinant genomes has been examined by restriction endonuclease analysis. The recombinants with a crossing over within or in the vicinity of the hexon gene were subjected to a more detailed analysis, using restriction enzyme maps of the HindIII A fragment (map coordinates, 50.1–72.8) constructed for endonucleases that cut the DNA many times, viz. HaeII, HindII, and TaqI. These analyses indicated that crossover sites are confined to regions of relatively high DNA homology. More detailed mapping within the hexon region of Ad2 and Ad5, using a large battery of enzymes, confirmed this and allowed subdivision into three zones on the basis of the distribution of restriction endonuclease recognition sites; at the left-hand amino terminus there is almost complete homology between Ad2 and Ad5, the right-hand half of the gene displays partial homology as judged by the even distribution of common and unique sites, while a central segment of the hexon gene has no common restriction sites. This segment, defined by unique sites, coincides with the region of the Ad2 hexon polypeptide implicated in the trypsin sensitivity of the native hexon, and probably also determines the type-specific antigenicity and electrophoretic mobility of the hexon.     

Tumorigenic poxviruses: analysis of viral DNA sequences implicated in the tumorigenicity of Shope fibroma virus and malignant rabbit virus

The DNA sequence has been determined for a 7-kb region within the terminal inverted repeats (TIR) of Shope fibroma virus (SFV), a poxvirus which induces benign fibromas in rabbits. This region of the SFV TIR, which flanks the junction of the TIR with the unique internal sequences of the viral genome, had previously been shown to be also present in the genome of malignant rabbit virus (MRV), a hybrid poxvirus derived from a recombination event between SFV and a related leporipoxvirus, myxoma. Unlike SFV, the recombinant MRV induces an invasive profile of tumors in infected rabbits, but the capacity to induce proliferant fibromas appears to have been derived from SFV. These SFV DNA sequences have been analyzed and their genetic organization shows a unique tandem arrangement of three large open reading frames (ORFs) which share considerable homology with each other. Very short spacer sequences are present between the majority of ORFs, all of which are transcribed toward the terminal hairpins of SFV. Unusual dyad symmetries flank two of the most closely related ORFs and evidence is presented that one SFV ORF (T9-L) which maps precisely at the TIR/unique sequence boundary was truncated during transposition to the left terminus from a progenitor copy (T9-R) at the right terminus. The origin of these putative viral genes is considered in light of the recent observation (C. Upton and G. McFadden, 1986, Mol. Cell. Biol. 6, 265-276) that a subset of this region of the SFV genome is closely related to, and may have been originally derived from, an endogenous covalently closed circular plasmid species detected in uninfected rabbit cells.     

Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6

The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 (EcoRI/Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 (PvuII CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the PvuII DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp and loop, 652 +/- 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.     

Use of λgt11 and monoclonal antibodies to map the gene for the 60,000 dalton glycoprotein of infectious laryngotracheitis virus

To localize the gene encoding the 60 kD glycoprotein (gp60) of infectious laryngotracheitis virus (ILTV), a library of the ILTV genome was constructed in the gt11 expression vector. Twelve recombinant bacteriophages expressing gp60 epitopes as fusion products with -galactosidase were detected by immunoscreening with monoclonal antibodies specific for gp60. The ILTV DNA sequence contained in one of these recombinants 24-4 was used as a hybridization probe for mapping the insert sequence on the viral genome. The gene for the gp60 was located at map unit 0.72–0.77 in the unique long region (U_L) of the ILTV genome. The DNA sequence of the 1.2 kb insert of 24-4 containing the gp60 epitope was determined. The majority of deduced gp60 amino acid sequence has no homology with any of the known alphaherpesvirus glycoproteins.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number X 121209.     

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.     

Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB,gC, gD,and gE

Summary We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.     

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface

The sequence of the 1491 nucleotides found at the 3' end of the genome of the highly aphid-transmissible (HAT) isolate of tobacco etch virus (TEV) has been determined. The nucleotide sequence of the capsid protein gene has been identified and compared with the corresponding region of the not-aphid-transmissible (NAT) isolate of TEV and with pepper mottle virus (PeMV). The deduced amino acid sequences of the two TEV capsid proteins displayed 98% homology and a 66% homology with PeMV capsid protein. Three of the six amino acid differences between the capsid proteins of the two TEV isolates occurred near the N terminus of the protein. Biochemical and immunological evidence suggested the N-terminal 29 amino acids of the capsid protein were hydrophilic and were located at or near the virion's surface.     

Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindIII J fragment despite divergent location of the thymidine kinase gene

DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.