Late transcripts of adenovirus type 12 DNA are not translated in hamster cells expressing the E1 region of adenovirus type 5.
(69/94)
Hamster cells are completely nonpermissive for the replication of human adenovirus type 12 (Ad12), whereas types 2 and 5 can replicate in hamster cells. The Ad5-transformed hamster cell line BHK297-C131, which carries the left terminal 18.7% of the Ad5 genome and expresses at least the viral E1A region, can somehow complement Ad12 DNA replication and the transcription of the late Ad12 genes. Since the interaction of Ad12 with hamster cells must constitute a significant factor in the induction of Ad12 tumors in neonatal hamsters, we have continued to examine details of this abortive virus infection. The late Ad12 mRNAs in BHK297-C131 cells are polyadenylated but are synthesized in reduced amounts compared with the Ad12 products in Ad12-infected human cells, which are permissive for viral replication. The late mRNA derived from the Ad12 fiber gene has been assessed for its structural properties. By cloning cDNA transcripts from this region and determining their nucleotide sequences, the authenticity of the complete Ad12 fiber sequence and the completeness of the Ad12-typical tripartite leader have been confirmed. Moreover, in Ad12-infected BHK297-C131 cells the Ad12 virus-associated RNA, a virus-encoded translational activator with the correct nucleotide sequence, is synthesized. Nevertheless, the synthesis of detectable amounts of Ad12 virion-specific proteins, and in particular that of the main viral antigens, hexons and fibers, cannot be documented. Cellular factors needed to promote late mRNA translation might be missing, or inhibitory factors might exist in Ad12-infected BHK297-C131 cells. (+info)
Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.
(70/94)
Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy. (+info)
Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit.
(71/94)
Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated. (+info)
Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.
(72/94)
Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins. (+info)