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

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

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

Arginine and glutamate rich 1 (ARGLU1) is a poorly understood cellular protein with functions in RNA splicing and transcription. Computational prediction suggests that ARGLU1 contains intrinsically disordered regions and lacks any known structural or functional domains. We used adenovirus Early protein 1A (E1A) to probe for critical regulators of important cellular pathways and identified ARGLU1 as a significant player in transcription and the DNA damage response pathway. Transcriptional effects induced by ARGLU1 occur via enhancement of promoter-proximal RNA polymerase II pausing, likely by inhibiting the interaction between JMJD6 and BRD4. When overexpressed, ARGLU1 increases the growth rate of cancer cells, while its knockdown leads to growth arrest. Significantly, overexpression of ARGLU1 increased cancer cell resistance to genotoxic drugs and promoted DNA damage repair. These results identify new roles for ARGLU1 in cancer cell survival and the DNA damage repair pathway, with potential clinical implications for chemotherapy resistance.

In Mitosis You Are Not: The NIMA Family of Kinases in Aspergillus, Yeast, and Mammals.

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.

Adenovirus 5 Vectors Expressing SARS-CoV-2 Proteins.

SARS-CoV-2 coronavirus is a recently identified novel coronavirus that is the causative agent of the COVID-19 pandemic that began in 2020. An intense research effort has been undertaken by the research community in order to better understand the molecular etiology of this virus and its mechanisms of host cell subjugation and immune system evasion. To facilitate further research into the SARS-CoV-2 coronavirus we have generated adenovirus 5-based viral vectors that express SARS-CoV-2 proteins-S, N, E, NSP7, NSP8, and NSP12 as hemagglutinin (HA)-tagged and untagged variants. We have also engineered two additional viruses that express the S protein receptor binding domain and a fusion of the receptor binding domain to the N protein. We show that these vectors are expressed in several different cell lines by Western blotting and real-time quantitative reverse transcriptase (qRT-PCR), we evaluate the subcellular localization of these viral proteins, and we show that these coronavirus proteins bind to a variety of cellular targets. The flexibility of adenovirus vectors allows them to be used in a variety of cell models and, importantly, in animal models as well. IMPORTANCE 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. Therefore, wide availability of resources and reagents to study SARS-CoV-2 is essential in overcoming the pandemic and for the prevention of future outbreaks. Our viral vectors provide additional tools for researchers to use in order to better understand the molecular biology of virus-host interactions and other aspects of SARS-CoV-2.

Differential Splicing of Human Adenovirus 5 E1A RNA Expressed in cis versus in trans.

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 a deletion of early region 1 (E1), 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 with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from 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 RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations 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 growth defect observed 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 similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the 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, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.

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

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.