Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape
© Imelli et al; licensee BioMed Central Ltd. 2009
Received: 12 June 2009
Accepted: 27 October 2009
Published: 27 October 2009
Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes.
Human adenoviruses (Ads) cause a wide range of diseases [1–3] but it is incompletely known how virus structure relates to infection. Ad particles consist of an icosahedral capsid enclosing a linear-double stranded DNA genome. The outer capsid is made of hexon (protein II), the penton base at the vertices (protein III), the protruding trimeric fibers (protein IV), and various minor proteins, IIIa, VI, VIII and IX. The inner core contains the double-stranded DNA with condensing proteins VII, V, and X, two copies of the terminal protein at the 5' ends of the DNA, the IVa2 core protein, and about 10 copies of the 23 kDa protease L3/p23. L3/p23 is highly conserved across human Ads, and has important roles in virion morphogenesis and entry . It cleaves substrates at glycine and isoleucine-containing consensus sites [5, 6], and requires cofactors for optimal activity [7–9]. During virion assembly, L3/p23 cleaves six structural precursor (p) proteins, pIIIa, pVI, pVII, pVIII, pX, the preterminal protein (pTP), and possibly the L1-52/55K scaffolding protein . L3/p23 cleaves V and pVII at putative cleavage sites, and hexon and pVI at degenerate cleavage sites in vitro.
The isolation of the temperature-sensitive (ts) Ad2-ts1 suggested that L3/p23 encoded a protease . Ad2-ts1 is defective in protease packaging, and virion processing at the nonpermissive temperature (40°C) . The mutation was mapped to P137L of L3/p23 , and eliminated by spontaneous reversions of the C22187T mutation in L3/p23 . Yet, the recombinant P137L protease is catalytically active [16, 17], and the ts1-phenotype rescued by adding a protease-activating peptide of the C-terminus of pVI to infected cells . It is unknown if secondary mutations in Ad2-ts1 act synergistically with P137L, and contribute to the phenotype.
To dissect the complex Ad2-ts1 entry phenotype [18, 19], we sequenced the structural proteins, the packaging-related proteins and the origins of replication of Ad2-ts1. Comparison of the Ad2-ts1 DNA sequence (GenBank accession numbers EU128936, EU128937, EU128938) to the Ad2 sequence (GenBank accession number AC_000007) revealed three mutations within the 3'untranslated regions (UTRs), and five mutations in the coding sequence (see Additional file 1). Two of the latter mutations were silent, and three affected the protein-coding sequences of Ad2-ts1, including C22187T (P137L substitution in L3/p23). G5043C in IVa2 gave rise to a H130D substitution, which was, however, strictly conserved among all other Ad sequences, and may represent an error in the original Ad2 GenBank entry. A deletion of three nucleotides in Ad2-ts1 protein V (GAT16677-16679) deleted D47. D47 was also missing in an Ad2 isolate (obtained from Dr. E. White), and Ad2-BAC53 which was generated from Ad2 "adenoid 6". Since D47 is not present in any known species C Ad sequences except the Genbank Ad2 published sequences, and is the last residue of a nonconserved stretch of five aspartate residues, we believe that the GAT triplet (16677-16679) in GenBank is a sequencing or entry error. It is unlikely that protein V contributes to the Ad2-ts1 phenotype, since viruses lacking protein V can be grown in cultured cells . We thus confirmed that the lack of proteolytic processing in Ad2-ts1 is not due to mutations in any of the protease consensus sequences.
The best-studied endocytic pathway is clathrin-mediated endocytosis. Clathrin-coated pits support transport of cargo between the plasma membrane, endosomes and the trans-Golgi-network (TGN) [33–35]. They are built around nucleating sites on membranes by adaptors and accessory proteins with multiple functions, including membrane bending and curvature sensing. Endocytic effector proteins like AP180 or CALM (clathrin assembly lymphoid myeloid) bind to both phosphatidylinositol 4,5-bisphosphate and clathrin . Overexpression of the carboxy-terminal clathrin heavy chain binding domain of AP180 (aa 530-915) prevents the recruitment of clathrin to the plasma membrane , and thereby inhibits clathrin-mediated endocytosis in many different cells types . It also inhibits Ad2 and Ad2-ts1 uptake into epithelial cells , supporting the notion that Ad2 and Ad2-ts1 enter by clathrin-mediated endocytosis [31, 39, 40].
This study provides new insights on how adenoviruses escape from endosomes. Both Ad2 and Ad2-ts1 attach to CAR, and use alpha v integrins for endocytic uptake [19, 31, 45]. Unlike Ad2, Ad2-ts1 fails to shed the fibers on the cell surface [24, 30]. We speculate that fiber shedding is critical for viral escape from endosomes either by involvement of penton base , or additional factors such as protein VI [45, 47]. Our results also provide a tool for genetic analyses of upstream events in clathrin-mediated endocytosis and membrane transport during Ad entry, and virion morphogenesis [48, 49]. For example, the P137L mutation of L3/p23 is located in a conserved surface-exposed loop, which may enable to generate Ad2-ts1-like mutants of other serotypes that fail to reach the cytosol, and do not trigger cytosolic DNA-sensing mechanisms in innate immunity [50, 51].
We thank Hans-Gerhard Burgert for Ad2-BAC53, and Karin Boucke for expert help with EM analyses. Funding was obtained from the Swiss National Science Foundation, the University of Zurich (to UFG), and the German Research Foundation (DFG SFB 455 to ZR). The authors declare that they have no competing interests.
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