Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization

Abstract

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression. IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

Differential Effects of Human Adenovirus E1A Protein Isoforms on Aerobic Glycolysis in A549 Human Lung Epithelial Cells

“…Low passage (<20) A549 cells with endogenous expression of the 13S encoded E1A isoform of HAdV-5 (A549-13S), the 12S encoded isoform of HAdV-5 E1A (A549-12S) or empty vector transduced control cells (A549-EV) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, L-glutamine, sodium pyruvate, and phenol red (Wisent Inc, Saint-Jean-Baptiste, QC, Canada) supplemented with 10% fetal bovine serum (Wisent Inc), and 1% penicillin-streptomycin (Wisent Inc). The A549-13S cells were previously described in Soriano et al [ 23 ] as A549-E1A289R cells. These cells and the A549-12S and A549-EV cells were generated concurrently as the A549-13S cells and in a similar manner.…”

Epigenetics and the dynamics of chromatin during adenovirus infections

“…Since E1A is known to interact with and modulate the activity of many host factors (see a comprehensive review by [83]), it is possible that the deletion of E1A residues 11-13 impacts more than its interaction with E4orf3. On viral genomes, transcription is reduced, histone occupancy increases, and E1A occupancy decreases only when E4orf3 is expressed in the presence of E1A compared to a control vector [143]. Based on these observations, E4orf3 seems to facilitate the association of host histone proteins with viral genomes and therefore modulate transcription.…”

“…A recent study suggests that E4orf3 may have a similarly repressive effect on transcription from viral genomes that is mediated through a heretofore unreported interaction between E4orf3 and E1A [143]. E1A co-immunoprecipitates with E4orf3 from cells that ectopically express an epitope-tagged copy of E4orf3 and are infected with E3D Ad5 demonstrating that E1A and E4orf3 interact during infection.…”

“…E1A co-immunoprecipitates with E4orf3 from cells that ectopically express an epitope-tagged copy of E4orf3 and are infected with E3D Ad5 demonstrating that E1A and E4orf3 interact during infection. On viral genomes, transcription is reduced, histone occupancy increases, and E1A occupancy decreases only when E4orf3 is expressed in the presence of E1A compared to a control vector [143]. Deletion analysis of E1A demonstrates that the N-terminal amino acid residues 11-13 are responsible for E1A's interaction with E4orf3.…”

Identification of human adenovirus replication inhibitors from a library of small molecules targeting cellular epigenetic regulators

“…We and others have shown that shortly after reaching the nucleus, HAdV DNA associates with histones and assembles into a chromatin-like structure similar to that of the host genome ( Giberson et al, 2012 , 2018 bib_Giberson_et_al_2012 bib_Giberson_et_al_2018 ; Komatsu and Nagata, 2012 ; Lynch et al, 2019 ; Ross et al, 2011 ; Wong et al, 2013 ). Virus-encoded genes are expressed from this template, and expression can be regulated by cellular epigenetic modifiers ( Hsu et al, 2018 ; Komatsu et al, 2011 ; Soriano et al, 2019 ). Although only a subset of cellular proteins involved in regulating the HAdV genome have been identified to date, these observations raise the possibility of targeting such proteins in order to inhibit viral gene expression and replication.…”