The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts.

Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multiprotein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-κB signaling, and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1-dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.

Fragile X-Related Protein FXR1 Controls Human Adenovirus Capsid mRNA Metabolism.

Human adenoviruses (HAdVs) are widespread pathogens causing a variety of diseases. A well-controlled expression of virus capsid mRNAs originating from the major late transcription unit (MLTU) is essential for forming the infectious virus progeny. However, regulation of the MLTU mRNA metabolism has mainly remained enigmatic. In this study, we show that the cellular RNA-binding protein FXR1 controls the stability of the HAdV-5 MLTU mRNAs, as depletion of FXR1 resulted in increased steady-state levels of MLTU mRNAs. Surprisingly, the lack of FXR1 reduced viral capsid protein accumulation and formation of the infectious virus progeny, indicating an opposing function of FXR1 in HAdV-5 infection. Further, the long FXR1 isoform interfered with MLTU mRNA translation, suggesting FXR1 isoform-specific functions in virus-infected cells. We also show that the FXR1 protein interacts with N6-methyladenosine (m6A)-modified MLTU mRNAs, thereby acting as a novel m6A reader protein in HAdV-5 infected cells. Collectively, our study identifies FXR1 as a regulator of MLTU mRNA metabolism in the lytic HAdV-5 life cycle. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, such as the common cold and conjunctivitis. Even though adenoviruses have been studied for more than 6 decades, there are still gaps in understanding how the virus interferes with the host cell to achieve efficient growth. In this study, we identified the cellular RNA-binding protein FXR1 as a factor manipulating the HAdV life cycle. We show that the FXR1 protein specifically interferes with mRNAs encoding essential viral capsid proteins. Since the lack of the FXR1 protein reduces virus growth, we propose that FXR1 can be considered a novel cellular proviral factor needed for efficient HAdV growth. Collectively, our study provides new detailed insights about the HAdV-host interactions, which might be helpful when developing countermeasures against pathogenic adenovirus infections and for improving adenovirus-based therapies.

The Human Adenovirus Type 2 Transcriptome: An Amazing Complexity of Alternatively Spliced mRNAs.

We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.

Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth.

The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A-cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.

An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication.

Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.

The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing.

The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells.

At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

Single molecule analysis of combinatorial splicing.

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.

ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression.

ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.

Ixovex-1, a novel oncolytic E1B-mutated adenovirus.

There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3' splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex.

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

Role of CCCH-Type Zinc Finger Proteins in Human Adenovirus Infections.

The zinc finger proteins make up a significant part of the proteome and perform a huge variety of functions in the cell. The CCCH-type zinc finger proteins have gained attention due to their unusual ability to interact with RNA and thereby control different steps of RNA metabolism. Since virus infections interfere with RNA metabolism, dynamic changes in the CCCH-type zinc finger proteins and virus replication are expected to happen. In the present review, we will discuss how three CCCH-type zinc finger proteins, ZC3H11A, MKRN1, and U2AF1, interfere with human adenovirus replication. We will summarize the functions of these three cellular proteins and focus on their potential pro- or anti-viral activities during a lytic human adenovirus infection.

Synthesis, Structure, and Function of Human Adenovirus Small Non-Coding RNAs.

Human adenoviruses (HAdVs) are common pathogens causing a variety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an advantage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV sncRNAs; VA RNA, mivaRNA and MLP-TSS-sRNA will be discussed in the present review. Even though VA RNA has been extensively characterized during the last 60 years, this multifunctional molecule continues to surprise us as more of its structural secrets unfold. Likely, the recent developments on mivaRNA and MLP-TSS-sRNA synthesis and function highlight the importance of these sncRNA in virus replication. Collectively, we will summarize the old and new knowledge about these three viral sncRNAs with focus on their synthesis, structure and functions.

Adenovirus VA RNAI Blocks ASC Oligomerization and Inhibits NLRP3 Inflammasome Activation.

Virus infected immune cells can rapidly respond to the invader by activating the inflammasome and as a consequence release proinflammatory cytokines and eventually die by pyroptosis. In human adenovirus-5 (Ad5) infected THP-1 cells, inhibition of NLRP3 inflammasome activation was demonstrated by a decreased secretion of HMGB1 and matured forms of caspase-1and IL-1ß. An Ad5 mutant virus defective in expression of the non-coding VA RNAI failed to inhibit the NLRP3 inflammasome and in addition displayed formation of ASC specks and increased cell lysis. Importantly, in vitro synthesized VA RNAI was able to inhibit the NLRP3 inflammasome activity in THP-1 cells in the absence of an Ad5 infection, suggesting that VA RNAI binding to PKR and blocking its function is sufficient for inhibition of the NLRP3 inflammasome. Although the inhibition of NLRP3 inflammasome activation required the phylogenetically conserved base paired tetranucleotide sequence in the central stem of VA RNAI, we demonstrate that PKR binding to VA RNAI primarily protected the apical stem, but not the tetranucleotide sequence itself. VA RNAI did not influence the interaction between PKR and NLRP3. In contrast, we describe a novel interaction between PKR and ASC and further show that VA RNAI inhibited ASC phosphorylation and oligomerization. Collectively, our results indicate a novel role for Ad5 VA RNAI as an inhibitor of NLRP3 inflammasome activation by targeting the cellular pro-inflammatory protein PKR.

Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression.

Effects of non-esterified fatty acids (FAs) are accentuated when applied together with elevated glucose through preferential use of glucose as fuel, which leads to decreased oxidation of FAs. We examined how over-expression of the mitochondrial FA transporter carnitine palmitoyltransferase 1 (CPT1) affects glucose-stimulated insulin secretion (GSIS), apoptosis and ER stress in INS-1E cells cultured in the presence of elevated levels of glucose and palmitate. INS-1E cells were infected with Tet-ON regulated adenovirus containing CPT1 and cultured for 48h in the presence of 0.5mM palmitate and 20mM glucose. Over-expressing CPT1 lowered basal insulin secretion in a dose-dependent manner thereby improving GSIS from INS-1E cells. Also, apoptosis was alleviated and ER-stress markers p-eIF2alpha and CHOP were decreased in cells over-expressing CPT1. We conclude that regulated over-expression of CPT1 is beneficial for glucolipotoxic beta-cells.

Temporal regulation of adenovirus major late alternative RNA splicing.

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

Preparation of soluble extracts from adenovirus-infected cells for studies of RNA splicing.

Here we describe a collection of methods that have been adapted to produce highly efficient nuclear and cytoplasmic extracts from adenovirus-infected HeLa cells. We describe how to produce extracts from virus-infected cells and how to analyze RNA splicing in vitro using T7 RNA polymerase-derived splicing substrate RNAs.

In vitro methods to study RNA interference during an adenovirus infection.

RNA interference (RNAi) has attracted a lot of interest during recent years as a method to knock-down gene expression and as a possible antiviral system. Here we present a collection of in vitro methods to study RNAi and the effect of an adenovirus infection on RNAi. We describe methods to measure the two key enzymatic complexes involved in RNAi: Dicer and RISC.

Expression profile of Epstein-Barr virus and human adenovirus small RNAs in tonsillar B and T lymphocytes.

We have used high-throughput small RNA sequencing to characterize viral small RNA expression in purified tonsillar B and T lymphocytes isolated from patients tested positive for Epstein-Barr virus (EBV) or human adenovirus (HAdV) infections, respectively. In the small set of patients analyzed, the expression profile of EBV and HAdV miRNAs could not distinguish between patients diagnosed with tonsillar hypertrophy or chronic/recurrent tonsillitis. The EBV miR-BART expression profile among the patients diagnosed with tonsillar diseases resembles most closely the pattern seen in EBV+ tumors (Latency II/I). The miR-BARTs that appear to be absent in normal EBV infected cells are essentially all detectable in the diseased tonsillar B lymphocytes. In the EBV+ B cells we detected 44 EBV miR-BARTs derived from the proposed BART precursor hairpins whereof five are not annotated in miRBase v21. One previously undetected miRNA, BART16b-5p, originates from the miR-BART16 precursor hairpin as an alternative 5´ miR-BART16 located precisely upstream of the annotated miR-BART16-5p. Further, our analysis revealed an extensive sequence variation among the EBV miRNAs with isomiRs having a constant 5´ end but alternative 3´ ends. A range of small RNAs was also detected from the terminal stem of the EBER RNAs and the 3´ part of v-snoRNA1. During a lytic HAdV infection in established cell lines the terminal stem of the viral non-coding VA RNAs are processed to highly abundant viral miRNAs (mivaRNAs). In contrast, mivaRNA expression in HAdV positive tonsillar T lymphocytes was very low. The small RNA profile further showed that the 5´ mivaRNA from VA RNAI and the 3´ mivaRNA from VA RNAII were as predicted, whereas the 3´ mivaRNA from VA RNAI showed an aberrant processing upstream of the expected Dicer cleavage site.