Characterization of Adenovirus 5 E1A Exon 1 Deletion Mutants in the Viral Replicative Cycle

Abstract

Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.

ARGLU1 enhances promoter-proximal pausing of RNA polymerase II and stimulates DNA damage repair

“…ARGLU1 was knocked-down via siRNA transfection followed by infection and determination of viral titer 24 and 48 h after infection with HAdV-C5 dl 309 (Figure 2E ). Depletion of ARGLU1 had an overall enhancing effect on virus growth, similar to what we previously observed with the mutant dl 1102 ( 43 ), with viral titers increasing approximately 2-fold with ARGLU1 knockdown versus siControl-treated cells. The enhanced virus growth correlated with enhanced viral gene expression when ARGLU1 was knocked down (Figure 2F ), similarly to what we previously have observed in E1A mutants that no longer bind ARGLU1 ( 43 ).…”

“…We have previously characterized the effects of deletion of E1A in the two mutants that have impaired binding to ARGLU1 ( dl 1102 and dl 1103) in primary lung fibroblasts WI-38 ( 43 ). Interestingly, we observed that mutant dl 1102 initially grew better than the in vitro phenotypically wild-type (wt) virus strain dl 309, with this mutant growing particularly well 48 and 72 h after infection and expressing its genes to higher levels than wt HAdV-C5, while mutant dl 1103 grew much worse than wt despite expressing its genes at a similar level to the wt virus ( 43 ). To determine whether knockdown of ARGLU1 affects viral replicative cycle in a similar way to deletion of its interaction region in E1A, we performed siRNA-mediated ARGLU1 knockdown studies.…”

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle

“…As previously described by Costa et al [ 47 ] and Graves et al [ 27 ], 293 cells were plated and then subsequently infected at a MOI 10 with either Ad.shHBO1 or Ad.shLuc in serum free media for 1 h. At the indicated time points, cells were imaged in the bright field at 20× magnification using the BioRad ZOE cell imager.…”

Adenovirus 5 Vectors Expressing SARS-CoV-2 Proteins

“…Real-time gene expression was performed as previously described ( 20 , 21 ). Briefly, 293 cells were infected in triplicate with each indicated viral construct at an MOI of 10 vp/cell for 24 h. Total RNA was extracted using TRI reagent (MilliporeSigma) according to the manufacturer’s instructions.…”