Genetic reconstitution of the human adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape.
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.
Institute of Zoology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Nicola
N
Imelli
Zsolt
Z
Ruzsics
Daniel
D
Puntener
Michele
M
Gastaldelli
Urs F
UF
Greber
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics*; D011992:Endosomes / Q000821:virology*; D020043:Genes, Essential; D006358:Hot Temperature; D006801:Humans; D020125:Mutation, Missense; D014764:Viral Proteins / Q000235:genetics*; D019065:Virus Assembly
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C082284:L3 23K protein, adenovirus