Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

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.

Direct Evidence from Single-Cell Analysis that Human α-Defensins Block Adenovirus Uncoating To Neutralize Infection

“…Having established conditions to produce labeled virus particles, we next sought to ensure that the immunofluorescence signal from BrdU was specific to uncoated virus in infected cells and that the labeling procedure did not alter the entry phenotype of the virus. One tool to aid these studies was a well-characterized HAdV-2 temperature sensitive mutant (HAdV-2ts1) that encodes a mutation in the viral protease gene (P137L) that greatly reduces the infectivity of virus grown at the nonpermissive temperature due to a failure to uncoat and escape the endosome (12,21,25). HAdV-2ts1 was produced under the same labeling conditions (4 g/ml BrdU) as wild-type HAdV-2 and was also found to contain labeled genomic DNA.…”

“…In order to use this mutant virus in immunofluorescence studies, we required a virus with the same phenotype as HAdV-2ts1 but with a HAdV-5 capsid to enable detection by a HAdV-5-specific antihexon MAb. To this end, we engineered the protease mutation P137L, which alone is responsible for the HAdV-2ts1 phenotype (12), into a HAdV-5-based vector expressing GFP. The high level of sequence conservation between HAdV-2 and HAdV-5 protease (99.5% identical amino acids) suggested that the phenotype of HAdV-5 bearing this mutation would be identical to that of HAdV-2ts1.…”

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

“…Virion motility in TC7 cells was determined by spinning disc confocal microscopy in the time window of 30-90 mpi. Cytosolic virions are the predominant entity in this time window, as indicated by previous studies showing that the half-time for virion penetration from endosomes is 15 min and that virions predominantly dock at the NPC after 30 min of infection Gastaldelli et al, 2008;Greber et al, 1997Greber et al, , 1993Greber and Way, 2006;Imelli et al, 2009;Meier et al, 2002;Suomalainen et al, 2013Suomalainen et al, , 2001Trotman et al, 2001). Movies with a length of 200 s were recorded at an image acquisition rate of 25 frames per second (Hz).…”

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

“…The temperature-sensitive human adenovirus serotype 2 mutant HAdV-C2-PRO-P137L (also termed ts1) was initially discovered in a chemical mutagenesis screen for growth-arrested viruses at the restrictive temperature of 38.5°C compared to the permissive temperature of 33°C (9). Subsequent analysis showed that the phenotype relies on a single point mutation in the AVP (P137L) that prevents virion incorporation of the AVP (8,10). Genomecontaining PRO-P137L capsids show a lack of proteolytic maturation, resulting in hyperstable capsids, presumably due to nonprocessed interactions between cement proteins and the viral core (11,12).…”