Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

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. Similar 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 integration.     

The CELO-ANG recombinant avian adenovirus with human angiogenine gene inducing neovascularization in the anterior tibial muscle of rat

The CELO recombinant avian adenovirus carrying the gene coding the human angiogenine (ANG) synthesis was obtained. Expression of the angiogenine gene was shown in the LMH cell culture after infection with the CELO-ANG virus. The ability of CELO recombinant adenoviruses to carry out the delivery and expression of alien genes in muscle cells was demonstrated in experiments with laboratory animals (Wistar line rats). The induced neovascularization in rat muscles after the animals were administered the CELO-ANG viruses was shown.     

Enhancement and inhibition of CELO virus pathogenicity in quail by avian adenovirus-associated virus

Dual infection of 12 day-old quail (Colinus virginianus) with 10(6) plaque forming units of CELO virus and low doses of avian adeno-associated virus (A-AV), resulted in significant enhancement of CELO virus-induced mortality, whereas dual infections with high doses of A-AV resulted in a delay in mortality. A-AV induced enhancement and inhibition of CELO virus pathogenicity could be blocked by the addition of A-AV antiserum prior to infection.     

Production of human recombinant beta-interferon in the avian cell culture

The avian recombinant adenovirus of serotype 1 (CELO) was obtained. The recombinant adenovirus of serotype 1 (CELO) induces expression of human beta-interferon (IB). The expression cassette containing IB gene was placed at the right end of the CELO genome under control of hybrid promoter hEF-1alpha/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of the recombinant IB was 0.15 micro/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was high (7.8 x 10(8) MU/microg protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, expression system based on avian cell culture is an effective system for producing biologically active protein of human interferon beta.     

Characterization of CELO Virus Proteins That Modulate the pRb/E2F Pathway

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.     

Construction of avian adenovirus CELO recombinants in cosmids

The avian adenovirus CELO is a promising vector for gene transfer applications. In order to study this potentiality, we developed an improved method for construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method, the ability to produce infectious particles and the stability of the genome were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). Our aim was to develop a replication-competent vector for vaccination of chickens, so we first generated knockout point mutations into 16 of the 22 unassigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs. An expression cassette coding for the enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then retained the vector backbone with the largest deletion (i.e., 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease virus proteins. These CELO vectors could be useful for vaccination in the chicken species.     

Eukaryotic vectors of Celo avian adenovirus genome,carrying GFP and human IL-2 genes

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.     

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

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 (Illa, 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 El, E3, and E4 functions.     

Mature Form of the Deoxyribonucleic Acid from Chick Embryo Lethal Orphan Virus

The deoxyribonucleic acid (DNA) of chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, had a biphasic denaturation profile indicating intramolecular base composition heterogeneity. This was confirmed by shearing the DNA and centrifuging it to equilibrium in Cs(2)SO(4) in the presence of HgCl(2) when two bands were formed. No circular molecules formed when CELO virus DNA was annealed, although lambda DNA formed circles under the same conditions. No circular molecules were found by sedimentation or electron microscopy when the DNA was digested with exonuclease III and then annealed, but 30 to 40% of T7 DNA molecules became circular under similar conditions. The complementary strands of CELO virus DNA both appeared to be continuous, and, when CELO DNA was denatured and then annealed under appropriate conditions, all of the renatured molecules were linear. It is concluded that CELO virus DNA consists of a unique rather than permuted collection of linear molecules that lack exposed single-strand complementary ends or duplex terminal repetitions. These results are discussed in relation to the replication of viral DNA and the transformation of host cells.     

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 A resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 (a=b=157.8 A, c=114.2 A, gamma=120 degrees) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large approximately 100 A diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.     

Comparison of Transformation and T Antigen Induction in Human Cell Lines

Skin fibroblast cultures were established from eight individuals. These cell cultures, together with WI-38 cells, were examined for susceptibility to transformation by SV40 virus. Four transformation-susceptible cell lines (TS), established from patients with Down's syndrome, were found to be three to four times more susceptible to transformation than transformation-resistant cell lines (TR) from normal individuals. TR and TS cell lines were compared for their susceptibility to induction of SV40 T antigen. For dividing cells T antigen was detected in a higher percentage of TS cells than TR cells. For nondividing cells, the reverse was found; T antigen was detected in 10-fold more cells of the TR lines than in cells of the TS lines. Similar results were obtained after infection of cells with CELO virus. Titration of vaccinia virus and influenza virus A2/Scotland/49/57 indicated that TR and TS cells were equally sensitive to the former virus, but TR cells were three to five times more sensitive to influenza virus A2/Scotland/49/57 than were TS cells.     

Cloning of terminal fragments of the avian adenovirus CELO genome and isolation of a recombinant plasmid carrying the viral oncogene

The terminal fragment of avian adenovirus CELO has been cloned in a plasmid vector. The obtained recombinant plasmid pCBE1 carries the terminal BamHI-E fragment of CELO DNA. Transfection of a nonpermissive culture of Rat2 cell line by the plasmid DNA results in formation of transformation focuses. The cloned BamHI-E fragment of CELO DNA is concluded to contain the viral oncogene. Thus, the CELO genome region deriving the BamHI-E fragment is "left".     

Influence of integrin-binding motif (RGD) on attachment and internalization of avian adenovirus CELO in mammalian cells

Recombinant avian adenovirus CELO bearing sequence RGD in the structure of a HI-loop of long fiber was designed. Experiments in vitro revealed that introduction of RGD-motif into fiber of CELO increased the ability of the virus to be attached to a surface of CAR-negative cells, and raised efficiency of the process of internalization of the virus both in CAR-positive, and in CAR-negative cells.     

Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs

Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.