Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have the early region 1 (E1) deleted, which is complemented by the cell line. Most commonly these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses deleted for E1 region often grow poorly on 293 cells when compared to E1 wild-type viruses. Therefore, we investigated whether this is caused by splicing differences between E1A provided by the cell line, or in trans; and that provided by the infecting viral genome, or in cis. We observed that E1A RNA that was expressed from the genome of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus, versus the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S that express E1A217R, E1A171R, and E1A55R isoforms respectively. We observed that these splicing differences likely arise due to different sub-nuclear localization of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell’s genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the observed growth defect with dl312 and resulted in higher virus growth.
IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various other similar cell lines are used extensively for virus production where high viral yields are important. Such complementing cell lines are used for production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans. Here we show that even complete genetic complementation of a viral gene does not result in full protein complementation, which compromises virus growth. This is particularly important where high viral yields are crucial, such as in virus production for vaccine development or gene therapy.
Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development
