Characterization of the recombinant adenovirus vector AdYB-1: implications for oncolytic vector development.

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.

Linked tumor-selective virus replication and transgene expression from E3-containing oncolytic adenoviruses.

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/DeltaE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/DeltaE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression.

Construction and characterization of E1-minus replication-defective adenovirus vectors that express E3 proteins from the E1 region.

Previous research has indicated that the adenovirus protein complex named RID, derived from the E3 transcription unit, functions to remove the receptors named Fas/Apo1/CD95 (Fas) and epidermal growth factor receptor (EGFR) from the surface of cells. (The RID complex is composed of the RIDalpha and RIDbeta polypeptides, previously named 10.4K and 14.5K, respectively.) In response to RID, Fas and EGFR appear to be internalized into endosomes and degraded in lysosomes. Fas is a death receptor in the tumor necrosis factor (TNF) receptor superfamily. RID inhibits apoptosis via the Fas pathway, presumably because RID gets rid of Fas. Earlier work further showed that another adenovirus E3-coded protein, E3-14.7K, inhibits apoptosis induced by TNF. Most of the above studies have been conducted using viable virus mutants that lack one or more of the genes for RID, E3-14.7K, or E1B-19K (this protein, coded by the E1B transcription unit, also inhibits apoptosis via the TNF and Fas pathways). Some studies have also been conducted with the genes for RID or E3-14.7K transiently or stably transfected into cells. We now report a new approach to studying the E3 genes. We have constructed four E1-minus replication-defective vectors that have all the E3 genes deleted from their natural position and then reinserted, in different permutations, into the deleted E1 region under control of the cytomegalovirus immediate early promoter. Vector Ad/RID only has the genes for RIDalpha and RIDbeta. Vector Ad/14.7K only has the gene for E3-14.7K. Vector Ad/RID/14.7K only has the genes for RIDalpha, RIDbeta, and E3-14.7K. Vector Ad/E3 has all E3 genes, but there are two missense mutations in the gene for Adenovirus Death Protein. These vectors expressed RID and/or E3-14.7K, as expected. The RID-expressing vectors forced the internalization and degradation of Fas and EGFR, and they inhibited apoptosis induced through the Fas pathway. These vectors should be useful reagents to study the E3 proteins.

Characterization of Ad5 E3-14.7K, an adenoviral inhibitor of apoptosis: structure, oligomeric state, and metal binding.

The adenovirus E3-14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF-alpha and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3-14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five beta-strands and one alpha-helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution.

E3-13.7 integral membrane proteins encoded by human adenoviruses alter epidermal growth factor receptor trafficking by interacting directly with receptors in early endosomes.

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7-mediated down-regulation in early endosomes. Receptor-viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675-697 are required for E3-13.7-mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

Early region 3 of adenovirus type 19 (subgroup D) encodes an HLA-binding protein distinct from that of subgroups B and C.

Early region 3 (E3) of human adenoviruses (Ads) codes for proteins that appear to control viral interactions with the host. For example, the most abundant E3 protein, E3/19K, inhibits the transport of newly synthesized class I major histocompatibility molecules to the cell surface, thereby interfering with antigen presentation. So far, the E3 regions of Ad subgroups A, B, C, and F have been characterized. We have cloned the E3A region of Ad type 19a (Ad19a), which belongs to the largest subgroup, D, and causes epidemic keratoconjunctivitis in humans. The sequence reveals five open reading frames (ORFs) with the potential to encode the Ad19 equivalent of pVIII, as well as proteins 12.2K, 16.2K, and 18.6K. The last ORF predicts a novel 49K protein which has no counterpart in other subgroups. Both the sequence and the overall organization of the E3 region from Ad19a shows a closer relationship to group B than to group C Ads. The 18.6K ORF represents the Ad19 homolog of the Ad2 E3/19K protein. By using 293 cells stably transfected with the Adl9a E3A region, we showed by immunoprecipitation, pulse-chase experiments, and fluorescence-activated cell sorter analysis that the Ad19 E3/19K protein binds to and prevents the transport of major histocompatibility complex molecules to the cell surface. The similar but distinct functional activity of the Ad19 E3/19K protein, combined with the new sequence which differs from those of subgroup B and C proteins, allows a more precise definition of amino acids essential for HLA binding.

The E3-20.5K membrane protein of subgroup B human adenoviruses contains O-linked and complex N-linked oligosaccharides.

Subgroup B adenoviruses (Ad3, -7, -11, -35) contain two open reading frames (ORFs) in the early E3 transcription unit that are not present in subgroup C adenoviruses (Ad2, Ad5). The product of one of these ORFs, a 20,500-kDa (20.5K) protein, was shown previously to be expressed as two diffuse 22K and 36K bands on SDS-PAGE; the 22K appeared to be the precursor to the 36K species. As judged by its predicted sequence, 20.5K is a type I membrane glycoprotein with two potential sites for N-glycosylation and a transmembrane domain near its COOH-terminus. Here we show that when Ad3- or Ad7-infected cells were radiolabeled in the presence of tunicamycin, which prevents the addition of N-linked oligosaccharides, both the 22K and the 36K forms of 20.5K showed increased mobility in SDS-PAGE, indicating that both forms contain N-linked sugars. Both the 22K and the 36K forms were sensitive to digestion by endoglycosidase F and N-glycanase, again indicating that they both contain N-linked sugars. Only the 22K species was sensitive to endoglycosidase H, indicating that it contains high-mannose-type oligosaccharides and that the 36K species contains complex-type carbohydrates. The 36K form was sensitive to neuraminidase, indicating that its sugars contain terminal sialic acid. When digested with N-glycanase and neuraminidase, the 36K form was sensitive to O-glycanase, indicating that the 36K form has O-linked oligosaccharides. The 22K form was labeled with [3H]mannose and the 36K form was labeled with [3H]glucosamine and to a much lesser extent by [3H]mannose. Altogether these results indicate that the 20.5K protein is cotranslationally modified with N-linked high-mannose oligosaccharides, then the protein moves into the Golgi and trans-Golgi network where it acquires O-linked and complex N-linked oligosaccharides.

Characterization of an 11K protein produced by early region 3 of mouse adenovirus type 1.

Early region 3 (E3) of mouse adenovirus type 1 produces three mRNAs that can encode three proteins with unique carboxy-terminal exons. A bacterial fusion protein encoding the unique terminus of one the three predicted proteins was used to generate antiserum in rabbits. This antiserum detected a 14K protein on a Western blot of infected cell lysates. Immunoprecipitation and endoglycosidase H digestion revealed that the 14K protein was a glycoprotein with a core molecular weight of 11K, and we are designating this protein E3 gp 11K. Through in vitro translation experiments we determined that the previously predicted signal sequence of gp11K was cleaved when the protein was expressed during an infection. Biochemical and immunofluorescence microscopy data indicated that E3 gp11K was localized to the endoplasmic reticulum of infected cells. Biochemical experiments further indicated that gp11K is a peripheral membrane protein.

The signal-anchor domain of adenovirus E3-6.7K, a type III integral membrane protein, can direct adenovirus E3-gp19K, a type I integral membrane protein, into the membrane of the endoplasmic reticulum.

Type III integral membrane proteins are oriented in the membrane with their C-terminus in the cytoplasm and their N-terminus extracytoplasmic. Such proteins are believed to have an internal hydrophobic sequence that functions both as an uncleaved signal for membrane insertion and also to anchor the protein in the membrane. However, type III proteins are relatively rare, and information about their putative signal-anchor (SA) domains is scant. The adenovirus E3-6.7K protein is a novel small type III protein. In order to study the insertion of 6.7K into membranes, we have constructed a fusion protein between 6.7K and adenovirus E3-gp19K; gp19K is a type I integral membrane protein that is known to form a complex with class I antigens of the major histocompatibility complex (MHC). The 6.7K-gp19K fusion protein lacks the gp19K signal sequence. We show that the 6.7K sequences can act as signal for membrane insertion of the 6.7K-gp19K fusion protein; however, the SA domain of 6.7K does not function as an anchor for the fusion protein. Thus, we have separated the signal function from the anchor function of the 6.7K SA domain. The transmembrane domain of gp19K is still acting as a stop-transfer sequence, and the ability of gp19K to bind MHC class I antigens is still intact. These data imply that sequences flanking a SA domain can influence whether the SA domain functions as a signal sequence only or as a dual signal-anchor sequence. The results also show that the signal for a type III membrane protein can direct a type I protein into the ER membrane. Finally, the data demonstrate that gp19K can retain its class I antigen binding function when gp19K has heterologous sequences fused to its N-terminus.