A simple and inexpensive method of condensing and linktransfection complexes deliver plasmid DNA to cells by the ing plasmid DNA to carrier adenovirus particles is adenovirus infectious route without interference from virus described. The synthetic polycation polyethylenimine is gene expression because psoralen-inactivated virus is used to condense plasmid DNA into positively charged 100 employed. The PEI-DNA-adenovirus complexes display nm complexes. These PEI-DNA complexes are then DNA delivery comparable to more sophisticated DNA virus bound to adenovirus particles through charge interactions complexes employing streptavidin/biotin linkage, but require with negative domains on the viral hexon. The resulting no special reagents and are much easier to prepare.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (IIIa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing E1, E3, and E4 functions.

A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.To successfully infect cells, adenoviruses must reach the nucleus, where viral gene expression can begin and the genomes can replicate. Adenoviruses are nonenveloped; the viral particle consists of an icosahedral protein capsid surrounding the 36-kb linear double-stranded DNA genome and associated DNA binding proteins (reviewed in reference 40). Human adenoviruses infect predominantly epithelial cells and can trigger respiratory and gastrointestinal tract ailments of a mild course in the majority of cases (reviewed in reference 23

Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent.

We have developed a simple screening method to identify genes that mimic bcl-2 or adenovirus E1B 19K in enhancing cell survival after transfection and have used this method to identify such a gene in the avian adenovirus CELO. The gene encodes a novel 30-kDa nuclear protein, which we have named GAM-1, that functions comparably to Bcl-2 and adenovirus E1B 19K in blocking apoptosis. However, GAM-1 has no sequence homology to Bcl-2, E1B 19K, or any other known antiapoptotic proteins and thus defines a novel antiapoptotic function. MATERIALS AND METHODS CELO library. CELO was grown and purified as previously described (14). CELO genomic DNA was prepared from purified CELO by using a sodium dodecyl sulfate-proteinase K digestion followed by banding in a CsCl density gradient. To create the pXCELO libraries, purified CELO DNA was digested with HindIII or EcoRI and cloned downstream from the cytomegalovirus (CMV) enhancer/promoter in plasmid pX (48). Individual plasmids bearing each of the expected fragments were isolated. Because of the structure of the adenovirus terminal fragments, the end fragments of the CELO genome were not obtained. The BssHII, SacII, SphI, and BglII GAM-1 mutations (see Fig. 3B) were prepared by digesting pX9R1SmaI/HindIII (which encodes GAM-1) with the indicated restriction enzyme, generating blunt ends by treatment with Klenow enzyme, and ligating. Plasmids bearing the expected mutations were isolated and verified by DNA sequencing. The resulting peptide sequences of the mutant proteins are presented in Table 1. The leucine mutations (L 258, 265 P; L 258, 265 A; and L 258, 265 G) were constructed using a PCR-based method; details can be supplied upon request. Additional plasmids. Plasmid pCMV-Bcl-2, encoding the bcl-2 cDNA (44) driven by the CMV promoter/enhancer, was provided by Michael Buschle (Institute for Molecular Pathology). The CMV-driven green fluorescent protein (GFP) expression plasmid pCMV-GFP was derived from the GFP cDNA derived by Chalfie et al. (7). The CMV-driven luciferase expression plasmid pCLuc was described earlier (40). pCMV-E1B 19K and pCMVE1A (59) were provided by Eileen White. For the fluorescence-activated cell sorting (FACS) analysis shown in Table 2, the enhanced GFP plasmid pEGFP-C1 (Clontech) was used. The NF-B-responsive luciferase reporter plasmid p3K-Luc was constructed as a derivative of pTK3kbB (2a). pTK3kbB was cleaved with XhoI and NcoI to remove most of the chloramphenicol acetyltransferase coding sequence, treated with Klenow enzyme, and ligated to a Klenow-treated HindIII/SspI fragment from pRSVL (15a) encoding the luciferase sequence. This resulted in plasmid p3K-Luc, containing a triple binding site for the transcription factor NF-B plus a thymidine kinase TATA box, driving the luciferase coding sequence. All plasmid preparations were purified and processed to remove lipopolysaccharide as previously described (12). Transfections. E4-defective adenovirus type 5 dl1014 (4) was grown on W162 cells (56), purified, and processed for biotinylation and 8-met...