Abstract
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.
Highlights
Human adenoviruses (Ads) cause a wide range of diseases [1,2,3] but it is incompletely known how virus structure relates to infection
transmission electron microscopy (TEM) analyses showed that the CALM knock-down cells contained less cytosolic Ad2 and more particles at the plasma membrane, but the distribution of Ad2-BAC46 particles was not significantly affected (p = 0.1, Fig. 3F, G)
CALM is involved in size regulation of clathrin-coated buds at the plasma membrane, its knockdown was reported not to affect internalization and recycling of transferrin, a well known ligand entering cells by clathrin-mediated endocytosis [42]
Summary
Human adenoviruses (Ads) cause a wide range of diseases [1,2,3] but it is incompletely known how virus structure relates to infection. TEM analyses showed that the CALM knock-down cells contained less cytosolic Ad2 and more particles at the plasma membrane, but the distribution of Ad2-BAC46 particles was not significantly affected (p = 0.1, Fig. 3F, G). Ad2-BAC46 infections (Fig. 3C-E), this suggests that CALM is involved in either uptake or endosomal escape of Ad2. CALM is involved in size regulation of clathrin-coated buds at the plasma membrane, its knockdown was reported not to affect internalization and recycling of transferrin, a well known ligand entering cells by clathrin-mediated endocytosis [42]. This could suggest http://www.virologyj.com/content/6/1/174 that Ad2-ts and Ad2-BAC46 follow an uptake pathway to early endosomes similar to transferrin. The P137L mutation of L3/p23 is located in a conserved surface-exposed loop, which may enable to generate Ad2ts1-like mutants of other serotypes that fail to reach the cytosol, and do not trigger cytosolic DNA-sensing mechanisms in innate immunity [50,51]
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