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The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

ABSTRACT: The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

SUBMITTER: Fleischli C

PROVIDER: S-EPMC1181579 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

Fleischli Christoph C Verhaagh Sandra S Havenga Menzo M Sirena Dominique D Schaffner Walter W Cattaneo Roberto R Greber Urs F UF Hemmi Silvio S

Journal of virology 20050801 15

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single ami ...[more]

PMID: 16014961

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Project description:BACKGROUND:Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS:Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS:Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION:Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.

Project description:Human granulocytes (polymorphonuclear leucocytes, PMN) possess a membrane cofactor protein (MCP, CD46), which is structurally and functionally distinct from the MCPs of other cell types: it shows a single broad band of 56-80 kDa (without the doublet pattern characteristic of MCP) on SDS/PAGE and has less affinity for complement component C3b. We purified PMN MCP using monoclonal antibodies in order to study the molecular differences between it and other MCPs. Several forms of PMN MCP with size heterogeneity were noted on SDS/PAGE and by immunoblotting. O-Glycanase treatment decreased this heterogeneity, yielding a fast-migrating component identical in position on SDS/PAGE to the O-glycanase-treated MCP of other cells. The cell-specific variation of MCP, therefore, arises from post-translational glycosylation and not from a difference in primary structure. The Factor I cofactor activity of PMN MCP was more efficient in cleaving the methylamine-treated complement components C4/C3 than was MCP from other cells, which shared a similar potency of cofactor activity on a weight basis. Two types of small-form PMN MCP were identified during purification. These were 42 kDa and 30 kDa in size; the former was recognized by M177 (a monoclonal antibody against the active site marker), possessed N-linked sugars [located on the short consensus repeats (SCRs)] but not O-linked ones (on the Ser/Thr-rich region), and retained cofactor activity for C3b/C4b cleavage, similar in potency to that of other MCPs. The functionally active soluble form of MCP was observed specifically in PMN. Protease inhibitors did not inhibit liberation of the fragments, although the generated fragments became susceptible to serine proteases. The findings show that the SCRs are the functional domain of MCP and that the MCP proteolysis found only in PMN may modulate the properties of PMN MCP. In conclusion, the structural features of PMN MCP largely reflect a variability in the O-linked sugars, and the decreased affinity for C3b may be in part attributable to proteolysis.

Dataset Information

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

Publications

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

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