IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation.

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.

Rb-E2F-HDAC Repressor Complexes Control Interferon-Induced Repression of Adenovirus To Promote Persistent Infection.

Interferons (IFNs) are cytokines that induce a global change in the cell to establish antiviral immunity. We previously demonstrated that human adenovirus (HAdV) exploits IFN-induced viral repression to persist in infected cells. Although this in vitro persistence model has been described, the mechanism behind how persistent HAdV infection is established is not well understood. In this study, we demonstrate that IFN signaling is essential for viral repression and promoting persistent infection. Cyclin-dependent kinase 4 (CDK4), an antagonist of retinoblastoma (Rb) family proteins, was shown to disrupt the viral repression induced by IFNs. Consistent with this result, knockout of the Rb family proteins pRb, p107, and/or p130 drastically reduced the effect of IFNs on viral replication. The pRb protein specifically contributed the greatest effect to IFN inhibition of viral replication. Interestingly, IFNs did not impact pRb through direct changes in protein or phosphorylation levels. Cells treated with IFNs continued to cycle normally, consistent with observations that persistently infected cells remain for long periods of time in the host and in our in vitro persistent infection model. Finally, we observed that histone deacetylase (HDAC) inhibitors activated productive viral replication in persistently infected cells in the presence of IFN. Thus, HDACs, specifically class I HDACs, which are commonly associated with Rb family proteins, play a major role in the maintenance of persistent HAdV infection in vitro. This study uncovers the critical role of pRb and class I HDACs in the IFN-induced formation of a repressor complex that promotes persistent HAdV infections. IMPORTANCE Adenoviruses are ubiquitous viruses infecting more than 90% of the human population. HAdVs cause persistent infections that may lead to serious complications in immunocompromised patients. Therefore, exploring how HAdVs establish persistent infections is critical for understanding viral reactivation in immunosuppressed individuals. The mechanism underlying HAdV persistence has not been fully explored. Here, we provide insight into the contributions of the host cell to IFN-mediated persistent HAdV infection. We found that HAdV-C5 productive infection is inhibited by an Rb-E2F-HDAC repressor complex. Treatment with HDAC inhibitors converted a persistent infection to a lytic infection. Our results suggest that this process involves the noncanonical regulation of Rb-E2F signaling. This study provides insight into a highly prevalent human pathogen, bringing a new level of complexity and understanding to the replicative cycle.

Cytokine pathway variants modulate platelet production: IFNA16 is a thrombocytosis susceptibility locus in humans.

Inflammatory stimuli have divergent effects on peripheral platelet counts, although the mechanisms of thrombocytopenic and thrombocytotic responses remain poorly understood. A candidate gene approach targeting 326 polymorphic genes enriched in thrombopoietic and cytokine signaling pathways was applied to identify single nucleotide variants (SNVs) implicated in enhanced platelet responses in cohorts with reactive thrombocytosis (RT) or essential (myeloproliferative neoplasm [MPN]) thrombocytosis (ET). Cytokine profiles incorporating a 15-member subset, pathway topology, and functional interactive networks were distinct between ET and RT, consistent with distinct regulatory pathways of exaggerated thrombopoiesis. Genetic studies using aggregate (ET + RT) or ET-restricted cohorts identified associations with 2 IFNA16 (interferon-α16) SNVs, and the ET associations were validated in a second independent cohort (P = .0002). Odds ratio of the combined ET cohort (n = 105) was 4.92, restricted to the JAK2V617F-negative subset (odds ratio, 5.01). ET substratification analysis by variant IFNA16 exhibited a statistically significant increase in IFN-α16 levels (P = .002) among 16 quantifiable cytokines. Recombinantly expressed variant IFN-α16 encompassing 3 linked non-synonymous SNVs (E65H95P133) retained comparable antiviral and pSTAT signaling profiles as native IFN-α16 (V65D95A133) or IFN-α2, although both native and variant IFN-α16 showed stage-restricted differences (compared with IFN-α2) of IFN-regulated genes in CD34+-stimulated megakaryocytes. These data implicate IFNA16 (IFN-α16 gene product) as a putative susceptibility locus (driver) within the broader disrupted cytokine network evident in MPNs, and they provide a framework for dissecting functional interactive networks regulating stress or MPN thrombopoiesis.

Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression.

Interferons (IFNs) are one of the hallmarks of host antiviral immunity. IFNs exert their antiviral activities through the induction of IFN-stimulated genes (ISGs) and antiviral proteins; however, the mechanism by which ISGs inhibit adenovirus (Ad) replication is not clearly understood. IFNs repress Ad immediate early gene expression and, consequently, all subsequent aspects of the viral life cycle. In this study, we found that IFN-induced protein with tetratricopeptide repeats 3, IFIT3 (ISG60), restricts Ad replication. IFIT3 repressed Ad E1A immediate early gene expression but did not alter Ad genome entry into the nucleus. Expression of IFIT3 led to phosphorylation of TBK1, IRF3, and STAT1; increased expression of IFNß and ISGs; and required IFIT1 and IFIT2 partner proteins. During RNA virus infections, it is known that IFIT3 stimulates IFN production through mitochondrial antiviral signaling (MAVS)-mediated activation of TBK1 which synergizes activation of IRF3 and NF-κB. MAVS or TBK1 depletion in cells expressing IFIT3 blocked IFN signaling and reversed the Ad replication restriction. In addition, STING depletion phenocopied the effect suggesting that IFIT3 activates the STING pathway with cross talk to the MAVS pathway. This occurs independently of viral pathogen-associated molecular patterns (PAMPs). These results demonstrate that the expression of a single ISG, IFIT3, activates IFN signaling and establishes a cellular antiviral state independent of viral PAMPs. IMPORTANCE IFITs belong to a family of IFN-induced proteins that have broad antiviral functions, primarily studied with RNA viruses leaving a gap of knowledge on the effects of these proteins on DNA viruses. In this study we show that IFIT3, with its partner proteins IFIT1 and IFIT2, specifically restricts replication of human Ad, a DNA virus, by stimulating IFNß production via the STING and MAVS pathways. This effect enhanced the IFN response and is independent of viral PAMPs. These results reveal a novel mechanism of activation of IFN signaling to enhance cellular antiviral responses.

Interferon-Lambda Intranasal Protection and Differential Sex Pathology in a Murine Model of SARS-CoV-2 Infection.

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.

NS5 Sumoylation Directs Nuclear Responses That Permit Zika Virus To Persistently Infect Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is cytopathic to neurons and persistently infects brain microvascular endothelial cells (hBMECs), which normally restrict viral access to neurons. Despite replicating in the cytoplasm, ZIKV and Dengue virus (DENV) polymerases, NS5 proteins, are predominantly trafficked to the nucleus. We found that a SUMO interaction motif in ZIKV and DENV NS5 proteins directs nuclear localization. However, ZIKV NS5 formed discrete punctate nuclear bodies (NBs), while DENV NS5 was uniformly dispersed in the nucleoplasm. Yet, mutating one DENV NS5 SUMO site (K546R) localized the NS5 mutant to discrete NBs, and NBs formed by the ZIKV NS5 SUMO mutant (K252R) were restructured into discrete protein complexes. In hBMECs, NBs formed by STAT2 and promyelocytic leukemia (PML) protein are present constitutively and enhance innate immunity. During ZIKV infection or NS5 expression, we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of interferon-stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression, or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 to regulate hBMEC cell cycle transcriptional responses. Our data reveal NS5 SUMO motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs.IMPORTANCE ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here, we demonstrate that flavivirus-specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization and that SUMO sites regulate NS5 NB complex constituents, assembly, and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs, and direct PML degradation. ZIKV NS5 SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons.

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry.

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.

The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections.

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.

Adenoviral strategies to overcome innate cellular responses to infection.

Viruses alter host cell processes to optimize their replication cycle. Human adenoviruses (Ad) encode proteins that promote viral macromolecular synthesis and counteract innate and adaptive responses to infection. The focus of this review is on how Ad evades innate cellular responses to infection, including an interferon (IFN) response and a DNA damage response (DDR). Ad blocks the IFN response by inhibiting cytoplasmic signaling pathways and the activation of IFN-stimulated genes (ISGs), as well as the functions of ISG products, such as PML. Ad also inhibits DDR sensors, for instance, the Mre11-Rad50-Nbs1 complex, and DDR effectors like DNA ligase IV. These innate cellular responses impact many different viruses, and studies on Ad have provided broad insight into these areas.

Adenovirus major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure.

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.

An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein.

The adenovirus (Ad) E4orf4 protein was reported to contribute to inhibition of ATM- and ATR-regulated DNA damage signaling during Ad infection and following treatment with DNA-damaging drugs. Inhibition of these pathways improved Ad replication, and when expressed alone, E4orf4 sensitized transformed cells to drug-induced toxicity. However, the mechanisms utilized were not identified. Here, we show that E4orf4 associates with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1) and that the association requires PARP activity. During Ad infection, PARP is activated, but its activity is not required for recruitment of either E4orf4 or PARP-1 to virus replication centers, suggesting that their association occurs following recruitment. Inhibition of PARP-1 assists E4orf4 in reducing DNA damage signaling during infection, and E4orf4 attenuates virus- and DNA damage-induced parylation. Furthermore, E4orf4 reduces PARP-1 phosphorylation on serine residues, which likely contributes to PARP-1 inhibition as phosphorylation of this enzyme was reported to enhance its activity. PARP-1 inhibition is important to Ad infection since treatment with a PARP inhibitor enhances replication efficiency. When E4orf4 is expressed alone, it associates with poly(ADP-ribose) (PAR) chains and is recruited to DNA damage sites in a PARP-1-dependent manner. This recruitment is required for inhibition of drug-induced ATR signaling by E4orf4 and for E4orf4-induced cancer cell death. Thus, the results presented here demonstrate a novel mechanism by which E4orf4 targets and inhibits DNA damage signaling through an association with PARP-1 for the benefit of the virus and impacting E4orf4-induced cancer cell death.IMPORTANCE Replication intermediates and ends of viral DNA genomes can be recognized by the cellular DNA damage response (DDR) network as DNA damage whose repair may lead to inhibition of virus replication. Therefore, many viruses evolved mechanisms to inhibit the DDR network. We have previously shown that the adenovirus (Ad) E4orf4 protein inhibits DDR signaling, but the mechanisms were not identified. Here, we describe an association of E4orf4 with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1). E4orf4 reduces phosphorylation of this enzyme and inhibits its activity. PARP-1 inhibition assists E4orf4 in reducing Ad-induced DDR signaling and improves the efficiency of virus replication. Furthermore, the ability of E4orf4, when expressed alone, to accumulate at DNA damage sites and to kill cancer cells is attenuated by chemical inhibition of PARP-1. Our results indicate that the E4orf4-PARP-1 interaction has an important role in Ad replication and in promotion of E4orf4-induced cancer-selective cell death.

Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK.

The adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCE Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization.

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression.IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications.

Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activities in vitro In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification. In vitro SUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation.IMPORTANCE Viruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occur in vivo We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9 in vitro only in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection.

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/µ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII-virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Adenovirus Early Proteins and Host Sumoylation.

The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system.

The Yersinia Virulence Factor YopM Hijacks Host Kinases to Inhibit Type III Effector-Triggered Activation of the Pyrin Inflammasome.

Pathogenic Yersinia, including Y. pestis, the agent of plague in humans, and Y. pseudotuberculosis, the related enteric pathogen, deliver virulence effectors into host cells via a prototypical type III secretion system to promote pathogenesis. These effectors, termed Yersinia outer proteins (Yops), modulate multiple host signaling responses. Studies in Y. pestis and Y. pseudotuberculosis have shown that YopM suppresses infection-induced inflammasome activation; however, the underlying molecular mechanism is largely unknown. Here we show that YopM specifically restricts the pyrin inflammasome, which is triggered by the RhoA-inactivating enzymatic activities of YopE and YopT, in Y. pseudotuberculosis-infected macrophages. The attenuation of a yopM mutant is fully reversed in pyrin knockout mice, demonstrating that YopM inhibits pyrin to promote virulence. Mechanistically, YopM recruits and activates the host kinases PRK1 and PRK2 to negatively regulate pyrin by phosphorylation. These results show how a virulence factor can hijack host kinases to inhibit effector-triggered pyrin inflammasome activation.