Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells.

Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts

Expression of cellular S-phase specific genes in infected IMR-90 cells.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. At the indicated time-points, total RNA was extracted using the TRIzol reagent and mRNA levels for the indicated genes were determined by qRT-PCR. GAPDH was used as a loading reference and mock-infected cells were used as a control and were set to 1. Error bars represent SD of 4 biological replicates. (B) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. One hour prior to the indicated time point cells were pulsed with EdU for 1 hour, fixed at the indicated time point, and stained for EdU and E1A as described in the Materials and Methods. Data represents percentage of infected cells that were positive for EdU staining. Error bars represent SD of 5 biological replicates.</p

Viral protein levels after infection of arrested IMR-90 cells.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>dl</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. At the indicated time-points, cells were lysed and 20μg total cellular lysate was resolved by SDS-PAGE on Novex BOLT 4–12% gradient minigel. E1A was detected with a combination of M58 and M73 monoclonal antibodies and visualized using a secondary HRP-conjugated anti-mouse antibody (Jackson Immunoresearch). (B) Same as A except probed for the 72kDa E2 DNA-binding protein. Secondary HRP-conjugated anti-mouse antibody (Jackson Immunoresearch) was used for detection. (C) Same as A except probed with antibody recognizing viral structural proteins (Abcam). Secondary anti-rabbit HRP-conjugated antibody was used for detection (Jackson Immunoresearch). (D) Same as A except probed for cellular actin (Abcam) as a loading control. Secondary anti-rabbit HRP-conjugated antibody was used for detection (Jackson Immunoresearch).</p

Virus growth and effects on cell morphology in arrested lung fibroblasts IMR-90.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. Virus titres were determined on 293 cells at the indicated time points. Inset shows E1A levels at 24 hours after infection. Error bars represent standard deviation (SD) of 4 replicate experiments. (B) Representative images of infected cells from A and mock-infected cells that were treated the same as infected cells, minus addition of virus. Images were taken prior to harvest of cells for titre determination and were taken at 100X magnification using phase-contrast optics.</p

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

Viruses alter a multitude of host-cell processes to create a more optimal environment for viral replication. This includes altering metabolism to provide adequate substrates and energy required for replication. Typically, viral infections induce a metabolic phenotype resembling the Warburg effect, with an upregulation of glycolysis and a concurrent decrease in cellular respiration. Human adenovirus (HAdV) has been observed to induce the Warburg effect, which can be partially attributed to the adenovirus protein early region 4, open reading frame 1 (E4orf1). E4orf1 regulates a multitude of host-cell processes to benefit viral replication and can influence cellular metabolism through the transcription factor avian myelocytomatosis viral oncogene homolog (MYC). However, E4orf1 does not explain the full extent of Warburg-like HAdV metabolic reprogramming, especially the accompanying decrease in cellular respiration. The HAdV protein early region 1A (E1A) also modulates the function of the infected cell to promote viral replication. E1A can interact with a wide variety of host-cell proteins, some of which have been shown to interact with metabolic enzymes independently of an interaction with E1A. To determine if the HAdV E1A proteins are responsible for reprogramming cell metabolism, we measured the extracellular acidification rate and oxygen consumption rate of A549 human lung epithelial cells with constitutive endogenous expression of either of the two major E1A isoforms. This was followed by the characterization of transcript levels for genes involved in glycolysis and cellular respiration, and related metabolic pathways. Cells expressing the 13S encoded E1A isoform had drastically increased baseline glycolysis and lower maximal cellular respiration than cells expressing the 12S encoded E1A isoform. Cells expressing the 13S encoded E1A isoform exhibited upregulated expression of glycolysis genes and downregulated expression of cellular respiration genes. However, tricarboxylic acid cycle genes were upregulated, resembling anaplerotic metabolism employed by certain cancers. Upregulation of glycolysis and tricarboxylic acid cycle genes was also apparent in IMR-90 human primary lung fibroblast cells infected with a HAdV-5 mutant virus that expressed the 13S, but not the 12S encoded E1A isoform. In conclusion, it appears that the two major isoforms of E1A differentially influence cellular glycolysis and oxidative phosphorylation and this is at least partially due to the altered regulation of mRNA expression for the genes in these pathways