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.

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.

The Never in mitosis gene A (NIMA) family of serine/threonine kinases is a diverse group of protein kinases implicated in a wide variety of cellular processes, including cilia regulation, microtubule dynamics, mitotic processes, cell growth, and DNA damage response. The founding member of this family was initially identified in Aspergillus and was found to play important roles in mitosis and cell division. The yeast family has one member each, Fin1p in fission yeast and Kin3p in budding yeast, also with functions in mitotic processes, but, overall, these are poorly studied kinases. The mammalian family, the main focus of this review, consists of 11 members named Nek1 to Nek11. With the exception of a few members, the functions of the mammalian Neks are poorly understood but appear to be quite diverse. Like the prototypical NIMA, many members appear to play important roles in mitosis and meiosis, but their functions in the cell go well beyond these well-established activities. In this review, we explore the roles of fungal and mammalian NIMA kinases and highlight the most recent findings in the field.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has brought untold personal and economic suffering to the world. Intense research has made tremendous progress in understanding how this virus works, yet much research remains to be done as new variants and continued evolution of the virus keep shifting the rules of engagement on the pandemic battlefield.