CELO virus: An oncogenic virus

198

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.

Section: Discussionmentioning

confidence: 99%

The complete DNA sequence and genomic organization of the avian adenovirus CELO

Chiocca

Kurzbauer

Schaffner

et al. 1996

198

“…We presumed that like most small DNA viruses, CELO must possess genes, such as those encoded by the E1A region, which influence cellular proliferation and genes such as E1B 55K and E1B 19K, which alter the cellular apoptotic response. Furthermore, CELO is capable of transforming cells and inducing tumors in newborn hamsters (1,27,32,33,43), demonstrating that the virus does possess transforming genes and could therefore possess functional homologs to the mastadenovirus E1A and E1B regions. Lacking an obvious sequence homolog, we have established a function assay to screen for a gene in CELO that slows the apoptotic response.…”

mentioning

confidence: 99%

Identification of a novel antiapoptotic protein, GAM-1, encoded by the CELO adenovirus

Chiocca

Baker

Cotten

1997

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...

“…CELO virus is an avian oncogenic adenovirus (22,23,32) able to transforn human and hamster cells in culture (1, 2, 28). In a previous study of CELO virus DNA replication in productively infected chicken embryo kidney (CEK) cells, we could not detect labeled parental viral DNA associated with cell DNA or newly synthesized viral DNA (labeled in a pulse of 1 h or less) sedirnenting faster than free viral DNA in alkaline sucrose gradients (4).…”

mentioning

confidence: 99%

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth

Tyndall¹,

Younghusband²,

Bellett³

1978

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Simiar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency of integration.