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

Abstract

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...

Characterization of a New Species of Adenovirus in Falcons

“…This and immunostaining for apoptotic DNA indicated that most tissue damage resulted from a direct cytolytic effect of virus rather than programmed cell death. The minor role of apoptosis is not surprising, since many adenoviruses, including FAV-1, have genes that encode antiapoptotic peptides (7). By contrast, HEV in turkeys causes intestinal tissue damage chiefly via apoptosis.…”

Viruses and sumoylation: recent highlights

“…Recent work from our laboratory has resulted in the publication of the first example of a viral protein, Gam1, that binds to the E1 heterodimer, inhibiting its function and causing a complete block of the sumoylation pathway both in vivo and in vitro [5]. Gam1 is an avian adenoviral protein that we have been focusing on as a model for the study of viral interference with cellular pathways [5][6][7][8][9][10].…”

Xenogenic Adenoviral Vectors

“…In FAdVl there are no identifiable ElA/B or E4 regions in the genome [23], but recently two proteins, GAM-1 and ORF22, that interact with pRb were identified [73]. In addition, GAM-1 has been identified as an antiapoptotic protein [111] and one that can activate the cellular heat-shock response, the latter being required for viral replication. The Hsp40 gene is a primary target [112].…”

“…The Hsp40 gene is a primary target [112]. GAM-1 may also functionally substitute for the ElB 19 kDa [111]. FAdVl therefore appears to share with the mastadenoviruses an ability to disrupt complexes between pRb and the E2F transcription factors to modulate the cell cycle, albeit via different effector proteins [99,113].…”

“…This may be more straightforward for the mastadenoviruses, where precedents exist from HAdV2/5 studies, but within this genus some viruses are more oncogenic than others [2,65] and some ORFs exhibit unexpected transforming properties [160][161][162]. Progress toward oncogene identification in FAdVl has also been made [73,111], but others may exist. Ultimately, the regulatory authorities will require tests to be conducted on the residual oncogenicity of xenogenic vectors prior to clinical application.…”