Left Inverted Terminal Repeat Research Articles

Recovery of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box motif (CACATG) deletion within the left terminal region

Muscovy duck-origin goose parvovirus (MDGPV) is a causative agent of MDGPV-associated Derzsy's disease. To evalute the role of the cis-acting element E-box (CACATG) deletion on MDGPV eplication, an infectious plasmid clone p-PTΔE287, having one E-box deletion at nucleotide (nt) 287 of the left inverted terminal repeat sequence (L-ITR), was constructed by overlap extension PCR deleting the 287CACATG292 motif from the plasmid pMDGPVPT containing the full-length genome of the virulent MDGPV strain PT. The p-PTΔE287 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac, resulting in successful rescue of the deletion mutant virus r-PTΔE287. Compared with its parental virus PT, the virulence and the replication ability of r-PTΔE287 were reduced. In addition, we examined the ability of r-PTΔE287 to manipulate cell cycle progression. The results showed that r-PTΔE287 replication results in G0/G1 phase accumulation of infected duck embryo liver mesenchymal stem cells (BMSCs) and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Taken together, introducing 287CACATG292 element deletion into MDGPV PT genomic DNA that induced rescued mutant virus (r-PTΔE287) cell cycle arrest function at the G0/G1 phase, which might inhibit MDGPV replication and virus progeny production. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.

Construction and rescue of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box deletion within the left terminal region

To obtain a deletion mutant of Muscovy duck-origin goose parvovirus (MDGPV) and to analyze its biological characteristics, the pMDGPVPT plasmid, which contains a full-length DNA infectious clone of the MDGPV PT strain, was used in this study as the template. The E-box at nt 315 of the left inverted terminal repeat sequence (L-ITR) was deleted by overlap extension PCR to obtain the infectious recombinant plasmid p-PTΔE315. The p-PTΔE315 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac and the rescued deletion mutant virus r-PTΔE315 was generated. Experiments to demonstrate the novel deletion mutant virus’ biological characteristics showed that r-PTΔE315 can cause typical lesions after infection of Muscovy duck embryos. Compared with its parent strain PT, the virulence of r-PTΔE315 and its proliferation ability in Muscovy duck embryos were attenuated, but its ability to replicate in MDEF cells was enhanced. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China.

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%–94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%–81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160–176 and 306–322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

Construction of a novel oncolytic adenoviral vector and its biological characteristics

In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the expression of HSV-1-TK gene making Ad-EITH sensitive to ACV. In conclusion, a novel oncolytic adenoviral vector Ad-EITH was produced which can be used for cancer-specific and efficient viral replication, and its safety is potentially improved as replication can be inhibited by ACV in vitro.

E1A promoter of bovine adenovirus type 3

Conserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR. As such, the promoter activity of ITR was tested in the context of a BAdV-3 vector or a plasmid-based system. Different regions of the left end of the BAdV-3 genome initiated transcription of the red fluorescent protein gene in a plasmid-based system. Moreover, BAdV-3 mutants in which the open reading frame of E1A was placed immediately downstream of the ITR produced E1A transcript and could be propagated in non-E1A-complementing Madin-Darby bovine kidney cells. These results suggest that the left ITR contains the sole BAdV-3 E1A promoter.

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication. Mutant PAdV-3s without the authentic E1A promoter region could be rescued by transfection of mutant genomic DNA into fetal porcine retina cells. Moreover, the mutant PAdV-3s produced E1A-specific mRNA and remained viable in swine testis (ST) cells suggesting that the left-terminal 151 bp including the ITR, can serve as a promoter for E1A expression. However, mutant PAdV-3s containing deletion including authentic E1A promoter region, displayed both reduced steady-state levels of early gene mRNAs (E1A, E1B, E2A, E3, and E4) and decreased rate of viral replication in ST cells. Interestingly, mutant PAdV-3s containing the left-terminal 312 bp displayed increased transcription of early genes including E1A. Our results suggest that the left ITR of PAdV-3 contain the promoter like elements and the sequences (between nt 151 and 312) downstream of left ITR can enhance its promoter activity.

Analyses of cis -acting elements that affect the transposition of Mos1 mariner transposons in vivo.

The left (5') inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3') ITR. The effects on the transposition frequency resulting from the use of two 3' ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3' ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3' ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3' ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.

Characterization of cis-acting sequences involved in packaging porcine adenovirus type 3

Encapsidation of adenovirus DNA involves specific interactions between cis-acting genomic DNA sequences and trans-acting proteins. The cis-acting packaging domain located near the left inverted terminal repeat is composed of a series of redundant but not functionally equivalent motifs. Such motifs are made up of the consensus sequence 5′-TTTGN 8CG-3′ and 5′-TTTG/A-3′ in human adenovirus 5 (HAV-5) and canine adenovirus-2 (CAV-2), respectively. To gain comparative insight into adenovirus encapsidation, we examined the packaging domain of porcine adenovirus-3 (PAV-3). Using deletion mutants, we localized the PAV-3 packaging domain to 319 bp (nt 212 to 531), which contains six cis-acting elements. However, this domain does not contain the consensus motifs identified in HAV-5. In addition, consensus motif found in CAV-2 is present only once in PAV-3. Instead, PAV-3 packaging domain appears to contain AT/GC-rich sequences. The packaging motifs of PAV-3, which are functionally redundant but not equivalent, are located at the left end of the genome.

A 72-bp Internal Deletion in the Left Inverted Terminal Repeat of the Bovine Adenovirus Type 3 Genome Does Not Affect Virus Replication

The genome of bovine adenovirus type 3 (BAV3) is flanked by 195-base pair (bp) inverted terminal repeats (ITR). We isolated a BAV3 mutant (BAV3c29) having an internal deletion within the left ITR. The deletion eliminated 72 bp between nucleotides (nt) 89 and 162, including most of the GC-rich sequences located close to the end of the ITR sequences. This deletion did not seem to have any affect on the virus plaque size or morphology and the kinetics of viral replication compared to wild-type (wt) BAV3. The nt sequence of the right ITR of BAV3c29 remained identical to the right or left ITR of wt BAV3. These results indicate that the cis-acting sequences present within the 72 bp between nt 89 and 162 of the left ITR are not essential for BAV3 DNA replication in cultured cells.

Characterization of cis-Acting Sequences Involved in Canine Adenovirus Packaging

The cis-acting packaging domain in adenovirus serotype 5 (Ad5) is a series of redundant, albeit not functionally equivalent, “A-repeats” made up of the consensus sequence 5′-TTTGN8CG-3′. A-repeats may bind trans-acting factors that direct packaging of the adenovirus genome into the preformed capsid. To try to understand this basic mechanism, we examined the packaging domain from a nonhuman adenovirus. We delimited the canine adenovirus type 2 (CAV-2) packaging domain to within 156 bp via a conditional mutation based on the Cre/loxP excision. Using an insertion, deletion, and substitution strategy, we generated packaging-defective CAV-2 vectors. Our results demonstrate that, like Ad5, CAV-2 cis-acting packaging sequences are located near the left inverted terminal repeat and are redundant, but not functionally equivalent. However, the bipartite motif found in Ad5 is present only once in CAV-2 and deletion of it caused only a minor variation in the packaging efficiency. We have identified at least four functional cis-acting packaging sequences in CAV-2. The CAV-2 vectors that we generated were not replication-defective in an E1-transcomplementing cell line and as heat stable as the parental vectors that did not contain mutations.

Sequence Analysis of Porcine Adenovirus Type 3 E1 Region, pIX and pIVa2 Genes, and Two Novel Open Reading Frames

The porcine adenovirus type 3 (PAd3) genome between map units 0 and 13.7 was sequenced and compared with similar regions of other adenoviruses. This region consists of the left inverted terminal repeat sequences involved in DNA packaging, the entire early region 1 (E1) and the protein IX (pIX) transcription unit. The lower strand contains the C-terminal end of IVa2 of the E2A transcriptional unit and two novel open reading frames (ORFs). The E1 transcription unit consists of ORFs for proteins homologous to the E1A, E1B-17k and E1B-55k of both human adenovirus type 5 (HAd5) and bovine adenovirus type 3 (BAd3). The predicted PAd3 pIX demonstrated homology with the N-terminal portion of the pIXs of HAd5 and BAd3. On the lower strand, immediately after the putative IVa2 ORF, there are two unique ORFs of 208 and 203 amino acid residues that showed homology with Epstein-Barr virus nuclear antigens and other cellular transcription factors.

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called P k1 containing most of the terminal repeat sequence was isolated and examined for promoter activity. P k1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn 903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative ( lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of P k1.The promoter activity of P k1 fragment was independent of the presence or absence of killer plasmids. It is not known whether P k1 can also function bidirectionally on the natural k1 plasmid. The possible functions of P k1 for killer plasmid gene expression and plasmid replication are discussed.

Multiple reiteration of a 40-bp nucleotide sequence in the inverted terminal repeat of the genome of a canine adenovirus

The DNA of a vaccine strain of canine adenovirus type 1 [ICHV vaccine; Connaught Laboratories, Ltd.; CAV-1(CLL)] has been cloned in plasmid pAT 153 in the form of subgenomic BamHI digestion fragments. Analysis of the nucleotide sequences of cloned terminal fragments has revealed an inverted terminal repeat (ITR) with a minimum lenght of 198 nucleotides, including a tandem reiteration of the 40-bp nucleotide sequence from positions 14 to 53. The ITRs had the 5′-CATCATCAAT … sequence typical of adenoviruses and the highly conserved sequence ATAATATAC (nucleotides 9–17) of human strains. Additionally, one BamHI A clone (left terminus) contained three sequential copies of the 40-bp sequence, and two BamHI C clones (right terminus) contained at least seven. These did not appear to be artifacts of cloning, since evidence was obtained that the multiple reiterations also occurred in DNA isolated from intact virus. By analogy with human adenoviruses, the repetitive sequence in the CAV-1(CLL) genome encompasses the entire nuclear factor I (NFI) binding site of the origin of DNA replication. Additionally, the 40-bp nucleotide sequence was found to contain the sequence AGG(N) 4GCCTAA (nucleotides 27–39), which closely resembles the concensus sequence of the human adenovirus NFI binding site [TGG(N) 6–7GCCAA; nucleotides 25–38]. It appears, therefore, that the Connaught CAV-1 vaccine contains reiterated copies of an essential part of the adenoviral origin of DNA replication. A mechanism is proposed for the generation of multiple reiterations of sequences in the right ITR, given an initial single tandem repeat in the left ITR.

Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

Site-specific nicking within the adenovirus inverted terminal repetition.

Site-specific nicking occurs on the l-strand, but not on the r-strand, of the adenovirus left inverted terminal repeat. Nicks are presumably introduced into double- or single-stranded DNA by a cellular endonuclease in an ATP-independent reaction. The consensus nick site has the sequence: (sequence in text).