Complex regulation of mitochondrial signaling by human adenovirus minor capsid protein VI.

The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.

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.

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.

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.

Distribution and Molecular Characterization of Human Adenovirus and Epstein-Barr Virus Infections in Tonsillar Lymphocytes Isolated from Patients Diagnosed with Tonsillar Diseases.

Surgically removed palatine tonsils provide a conveniently accessible source of T and B lymphocytes to study the interplay between foreign pathogens and the host immune system. In this study we have characterised the distribution of human adenovirus (HAdV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in purified tonsillar T and B cell-enriched fractions isolated from three patient age groups diagnosed with tonsillar hypertrophy and chronic/recurrent tonsillitis. HAdV DNA was detected in 93 out of 111 patients (84%), while EBV DNA was detected in 58 patients (52%). The most abundant adenovirus type was HAdV-5 (68%). None of the patients were positive for HCMV. Furthermore, 43 patients (39%) showed a co-infection of HAdV and EBV. The majority of young patients diagnosed with tonsillar hypertrophy were positive for HAdV, whereas all adult patients diagnosed with chronic/recurrent tonsillitis were positive for either HAdV or EBV. Most of the tonsils from patients diagnosed with either tonsillar hypertrophy or chronic/recurrent tonsillitis showed a higher HAdV DNA copy number in T compared to B cell-enriched fraction. Interestingly, in the majority of the tonsils from patients with chronic/recurrent tonsillitis HAdV DNA was detected in T cells only, whereas hypertrophic tonsils demonstrated HAdV DNA in both T and B cell-enriched fractions. In contrast, the majority of EBV positive tonsils revealed a preference for EBV DNA accumulation in the B cell-enriched fraction compared to T cell fraction irrespective of the patients' age.

HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice.

Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term "tonsils" refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Poor growth of human adenovirus-12 compared to adenovirus-2 correlates with a failure to impair PKR activation during the late phase of infection.

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-ß expression was transient, HAdV-12-infected cells maintained high levels of IFN-ß expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections.

A mouse mammary epithelial cell line permissive for highly efficient human adenovirus growth.

Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.

Activation of the interferon-induced STAT pathway during an adenovirus type 12 infection.

We have previously described a temporal regulation of host cell gene expression during adenovirus type 2 infection (Ad2) of primary human fibroblasts. Among the eleven percent of genes deregulated by Ad2, a large fraction included genes involved in cell cycle, growth control and antiviral defense, consistent with the capacity of Ad2 to efficiently master the infected cell and cause an effectively productive infection. Adenovirus type 12 (Ad12), which belongs to the highly oncogenic subgroup, is characterised by slow progression, less cytopathic effect and lower virus yield compared to the non-oncogenic Ad2. Microarray analysis of host cell gene expression in Ad12 infected human lung fibroblasts (IMR90) demonstrated a quantitatively and qualitatively less impact on host cell gene expression, compared to Ad2. Of the relatively few genes up regulated during the course of Ad12 infection only two (5%) were identified as potential E2F targets, compared to the significant activation of E2F-dependent transcription observed during an Ad2 infection. Although approximately 30% of the genes deregulated by Ad12 were previously identified in Ad2-infected cells, a distinct difference was observed in a group of interferon-stimulated genes (ISGs). G1P2, IFI6, IFI16, IFIT1, IFIT2, IFITM1 and IRF9 were activated during the very late stage of infection, and a consistent induction of IFNbeta gene expression, preceding induction of the ISGs, was demonstrated by quantitative real-time PCR analysis. An activated JAK/STAT signalling pathway was also indicated by the accumulation of all components (STAT1, STAT2 and IRF9) of the ISGF3 transcription factor. Significantly, none of these ISGs was activated in Ad2 infected IMR90 cells. Thus, the inability of Ad12 to evade the interferon response might explain its restricted virulence.

Adenovirus-induced alterations in host cell gene expression prior to the onset of viral gene expression.

In this report, we have studied gene expression profiles in human primary lung fibroblasts (IMR-90) during the very early phase of an adenovirus infection. Eight out of twelve genes with known functions encoded transcription factors linked to two major cellular processes; inhibition of cell growth (ATF3, ATF4, KLF4, KLF6 and ELK3) and immune response (NR4A1 and CEBPB), indicating that the earliest consequences of an adenovirus infection are growth arrest and induction of an immune response. A time course analysis showed that the induction of these immediate-early response genes was transient and suppressed after the onset of the adenovirus early gene expression.

Modulation of host cell gene expression during onset of the late phase of an adenovirus infection is focused on growth inhibition and cell architecture.

Microarray analysis of host cell gene expression during an adenovirus type 2 infection showed that the number of regulated genes, as well as the magnitude of change, was increased as the infection proceeded into the late phase. In contrast to the early phase of infection when the majority of differentially expressed genes were upregulated, expression of most of the regulated genes (82 out of 112) declined during the late phase. In particular, numerous TGF-beta inducible genes and several TGF-beta-independent growth-arrest-inducing genes were targeted. Of the 30 genes upregulated more than 2-fold at 20 h post-infection, nearly two-thirds of encoded proteins are involved in cell metabolism. The data indicate that adenovirus primarily targets cellular genes involved in antiviral defense, cell growth arrest and apoptosis, as well as cell metabolism, to ensure sufficient production of viral progeny.

The hTERT and hTERC telomerase gene promoters are activated by the second exon of the adenoviral protein, E1A, identifying the transcriptional corepressor CtBP as a potential repressor of both genes.

Telomerase plays a role in the unlimited replicative capacity of the majority of cancer cells and provides a potential anticancer target. The regulation of telomerase is complex but transcriptional control of its two essential components, hTERC (RNA component) and hTERT (reverse transcriptase component), is of major importance. To investigate this further, we have used the adenoviral protein, E1A, as a tool to probe potential pathways involved in the control of telomerase transcription. The second exon of the adenoviral protein E1A activates both telomerase gene promoters in transient transfections. The corepressor, C terminal binding protein, is one of only two proteins known to bind to this region, and we propose that E1A activates both promoters by sequestering CtBP, thereby relieving repression. Activation by exon 2 of E1A involves the SP1 sites in both promoters, and consistent with this, SP1 and CtBP interact in coimmunoprecipitation studies. Modulation of the chromatin environment has been implicated in the regulation of hTERT transcription and appears to involve the SP1 sites. CtBP can be found within a histone-modifying complex and it is possible that a CtBP complex, associating with the SP1 sites, represses transcription from the telomerase promoters by modifying chromatin structure.

Impact of the interaction between adenovirus E1A and CtBP on host cell gene expression.

In cell lines harbouring inducible adenovirus E1A genes, the cytotoxicity of wild type E1A was manifested by poor and subsiding expression of the E1A protein during prolonged induction. In contrast, cells expressing E1A deleted in the C-terminal binding protein (CtBP)-interaction domain (E1ADeltaCID) demonstrated high levels of expression for extended time. Microarray analyses of host cell gene expression demonstrated that approximately 70% of the regulated genes were increased upon E1A induction and that the majority of E1A-regulated genes were similarly regulated by wild type E1A and E1ADeltaCID. However, for 29 genes, regulation by wild type E1A and E1ADeltaCID were different. Consistent with the altered transforming capacity of E1A unable to bind CtBP, genes involved in tumour cell progression and growth suppression were found among the differently regulated genes. Moreover, promoter sequences of genes up regulated by wild type E1A and/or repressed by E1ADeltaCID demonstrated a higher prevalence of potential binding sites for the CtBP-targeted transcription factors Ets, Ikaros and/or partial differentialEF1/ZEB, suggesting that the failure to block CtBP-repression contributed to the "hyper-transforming" phenotype of E1ADeltaCID. Since E1ADeltaCID also specifically activated host cell gene expression, we find it likely that additional, possibly CtBP-independent, mechanisms contribute to the altered phenotype of E1ADeltaCID-expressing cells.

Identification of specific cellular genes up-regulated late in adenovirus type 12 infection.

The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.

Strategic attack on host cell gene expression during adenovirus infection.

To understand the interaction between the virus and its host, we used three sources of cDNA microarrays to examine the expression of 12,309 unique genes at 6 h postinfection of HeLa cells with high multiplicities of adenovirus type 2. Seventy-six genes with significantly changed expression ratios were identified, suggesting that adenovirus only modulates expression of a limited set of cellular genes. Quantitative real-time PCR analyses on selected genes were performed to confirm the microarray results. Significantly, a pronounced transcriptional activation by the promiscuous E1A-289R transcriptional activator was not apparent. Instead, promoter sequences in 45% of the upregulated genes harbored a potential E2F binding site, suggesting that the ability of the amino-terminal domain of E1A to regulate E2F-dependent transcription may be a major pathway for regulation of cellular gene expression. CDC25A was the only upregulated gene directly involved in cell cycle control. In contrast, several genes implicated in cell growth arrest were repressed. The transforming growth factor beta superfamily was specifically affected in the expression of both the upstream ligand and an intracellular regulator. In agreement with previous reports, adenovirus also targeted the innate immune response by downregulating several cytokines, including CLL2, CXCL1, and interleukin-6. Finally, stress response genes encoding GADD45B, ATF3, and TP53AP1 were upregulated. Importantly, we also found a novel countermeasure-activation of the apoptosis inhibitor survivin.