Reduced Infectivity of Adenovirus Type 5 Particles and Degradation of Entering Viral Genomes Associated with Incomplete Processing of the Preterminal Protein

Abstract

To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064 -3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells. Successful initiation of the human adenovirus infectious cycle depends on a complex set of interactions among viral and cellular components that allow attachment, entry, and partial dismantling of virus particles prior to the transport of viral genomes to and into the infected cell nucleus. The nonenveloped, icosahedrally symmetric virus particles carry distinctive fibers that project from the penton base present at each of the 12 vertices (5, 72). The distal knob of the fiber contains the binding site for attachment to the primary cell surface receptor, the coxsackievirus and adenovirus receptor, Car, in the case of species C adenoviruses such as serotype 5 (Ad5) (4,70,89). Interactions of RGD sequences present in loops that project from the surface of each subunit of the pentameric penton base with ␣v integrins on the cell surface (14, 84) then promote the entry of virus particles by clathrin-mediated endocytosis (18,50,75,80,101; reviewed in reference 1). Subsequent escape from early endosomes into the cytoplasm is coordinated with, and dependent on, initial uncoating reactions that remove capsid proteins.

Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

“…To avoid failure to capture proteins that interact with the region of the E1B 55 kDa protein containing this epitope, we attempted to construct vectors for expression of tagged E1B proteins in cells infected by mutant viruses defective for synthesis of only this viral protein, such as AdEasyE1Δ2347 (Kato et al, 2012). However, addition of coding sequences specifying, for example, a FLAG tag resulted in plasmid sequence loss in E. coli (N-terminal tag) or prevented the localization of the tagged E1B protein to the nucleus in infected cells (C-terminal tag) (C.J.…”

The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication

“…Indeed, replication of Hr6 in normal human fibroblasts or epithelial cells was observed to be several orders of magnitude more sensitive to exposure of cells to IFN than replication of Ad5 (46). The identical phenotype was exhibited by an E1B 55-kDa protein null mutant, AdEasyE1⌬2347, engineered to contain the Hr6 mutation (deletion of bp 2347 [48]), which prevents production of the E1B 55-kDa protein, but none of the other mutations recently identified in the Hr6 genome (49). Furthermore, the concentrations of pre-mRNAs synthesized from several IFN-inducible genes were increased substantially in cells infected by Hr6 and AdEasyE1⌬2347 compared to those in cells infected by the wild-type parental viruses in the absence or presence of exogenous IFN (46).…”

Human Adenovirus Type 55 Distribution, Regional Persistence, and Genetic Variability

“…Earlier work by Hay et al (47,48) demonstrated that point mutations and deletions generated on pTP affect DNA replication activity in vitro. Flint et al (49) showed that G315V substitution in pTP of HAdV-5 impaired pTP maturation leading to reduced infectivity. The remaining genomic changes of HAdV-55 appear to be largely trivial and not likely responsible Figure 2.…”