Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

Correction: ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1003632.].

Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle.

UNLABELLED: Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1-RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle. IMPORTANCE: Although influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA polymerase II, and with chromatin remodelers, such as CHD6. We identified the association of viral polymerase with another chromatin remodeler, the CHD1 protein, which positively modulated viral polymerase activity, viral RNA transcription, and virus multiplication. Once viral transcription is complete, RNAP II is degraded in infected cells, probably as a virus-induced mechanism to reduce the antiviral response. CHD1 associated with RNAP II and paralleled its degradation during infection with viruses that induce full or reduced degradation. These findings suggest that RNAP II degradation and CHD1 degradation cooperate to reduce the antiviral response.

Potential drug-drug interactions in hospitalised haematological patients.

Background Frequently, haematological patients undergo highly complex and intensive treatment protocols, so a high risk of drug-drug interactions could be expected. Objectives To determine prevalence of clinically relevant drug-drug interactions, to identify the most frequent drug-drug interactions and associated risk factors. Methods A prospective, observational and descriptive study was carried out from November 2012 to February 2013. Twice a week, every patient's treatment sheet was collected. Each medication list was screened through two databases: Thomson MicromedexTM and Drug Interaction FactsTM. All identified potential drug-drug interactions with a moderate or higher severity rating were recorded. Summary statistics were used to describe patient and disease characteristics, most often prescribed drugs, and frequency, types and classification of drug-drug interactions. Multiple logistic regression models were used to identify risk factors associated with drug-drug interactions. Results A total of 2061 drug-drug interactions were detected in 317 treatment sheets from 58 patients. The prevalence of treatment sheets with drug-drug interactions by Micromedex and Drug Interaction Facts databases were 74.1% and 56.8%, respectively. Azole antifungals, immunosuppressive drugs, antiemetics, antidepressants, acid suppressants and corticosteroids were the most frequent involved drugs. In multivariate analysis, the main risk factor associated with increased odds for drug-drug interactions was a higher number of non-antineoplastic drugs. Conclusions The prevalence of drug-drug interactions was common, with immunosuppressant and azole antifungal agents being the most commonly involved drugs. The factor having the greatest influence on drug-drug interactions was a higher number of non-antineoplastic drugs.

The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication.

UNLABELLED: Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. IMPORTANCE: Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member of the Microrchidia family that is associated with the nuclear matrix, during virus infection. NXP2/MORC3 associates with the viral ribonucleoproteins in infected cells. Downregulation of NXP2/MORC3 reduced virus titers and accumulations of viral genomic RNA and mRNAs. Silencing of NXP2/MORC3 in an influenza virus CAT minireplicon system diminished CAT protein and cat mRNA levels but not genomic RNA levels. We propose that NXP2/MORC3 plays a role in influenza virus transcription.

Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

UNLABELLED: Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. IMPORTANCE: Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.

ISG15 regulates peritoneal macrophages functionality against viral infection.

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15-/- macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.

Clinical response to pandemic H1N1 influenza virus from a fatal and mild case in ferrets.

BACKGROUND: The majority of pandemic 2009 H1N1 (A(H1N1)pdm09) influenza virus (IV) caused mild symptoms in most infected patients, however, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. The purpose of this work was to study in ferrets the dynamics of infection of two contemporary strains of human A(H1N1)pdm09 IV, one isolated from a patient showing mild disease and the other one from a fatal case. METHODS: Viral strains isolated from a patient showing mild disease-M (A/CastillaLaMancha/RR5661/2009) or from a fatal case-F (A/CastillaLaMancha/RR5911/2009), both without known comorbid conditions, were inoculated in two groups of ferrets and clinical and pathological conditions were analysed. RESULTS: Mild to severe clinical symptoms were observed in animals from both groups. A clinical score distribution was applied in which ferrets with mild clinical signs were distributed on a non-severe group (NS) and ferrets with severe clinical signs on a severe group (S), regardless of the virus used in the infection. Animals on S showed a significant decrease in body weight compared to animals on NS at 4 to 7 days post-infection (dpi). Clinical progress correlated with histopathological findings. Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) increased on both groups after 2 dpi. Clinically severe infected ferrets showed a stronger antibody response and higher viral titres after infection (p = 0.001). CONCLUSIONS: The severity in the progress of infection was independent from the virus used for infection suggesting that the host immune response was determinant in the outcome of the infection. The diversity observed in ferrets mimicked the variability found in the human population.

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin.

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

CHD6, a cellular repressor of influenza virus replication, is degraded in human alveolar epithelial cells and mice lungs during infection.

The influenza virus polymerase associates to an important number of transcription-related proteins, including the largest subunit of the RNA polymerase II complex (RNAP II). Despite this association, degradation of the RNAP II takes place in the infected cells once viral transcription is completed. We have previously shown that the chromatin remodeler CHD6 protein interacts with the influenza virus polymerase complex, represses viral replication, and relocalizes to inactive chromatin during influenza virus infection. In this paper, we report that CHD6 acts as a negative modulator of the influenza virus polymerase activity and is also subjected to degradation through a process that includes the following characteristics: (i) the cellular proteasome is not implicated, (ii) the sole expression of the three viral polymerase subunits from its cloned cDNAs is sufficient to induce proteolysis, and (iii) degradation is also observed in vivo in lungs of infected mice and correlates with the increase of viral titers in the lungs. Collectively, the data indicate that CHD6 degradation is a general effect exerted by influenza A viruses and suggest that this viral repressor may play an important inhibitory role since degradation and accumulation into inactive chromatin occur during the infection.

[Age group, geographical incidence and patterns of antifungal susceptibility of Candida species causing candidemia in the Spanish paediatric population]. / Incidencia etaria y geográfica y patrón de sensibilidad a los antifúngicos de las especies de Candida causantes de candidemia en la población pediátrica española.

INTRODUCTION: There are few epidemiological studies on candidaemia in the paediatric population in Spain. We sought to determine the epidemiology of candidaemia in these patients. METHODS: Prospective, observational and multicentre study in 44 Spanish hospitals. All candidaemia episodes in paediatric patients from 0 to 15 years old between January 2009 and February 2010 were studied. RESULTS: There were 197 episodes and 200 species were isolated. The most frequent species was Candida parapsilosis sensu stricto (43%), followed by C. albicans (36%), C. tropicalis (6%), C. orthopsilosis, and C. glabrata (4%) respectively. C. albicans was the most prevalent in newborns, and C. parapsilosis was most frequent in the other age groups. As regards the regions of Spain, C. albicans was most prevalent in patients from Catalonia, the Balearic Islands and Canary Islands, and C. parapsilosis in patients from Andalusia, Castilla-León, Galicia, Valencia, and Madrid. The rate of resistance to fluconazole was 1.5% (4.1% with the new species-specific Clinical and Laboratory Standards Institute [CLSI] criteria). Fluconazole resistance was lower in neonates than the other age groups. The Neonatal Wards were the areas with most episodes (31.5%). In the multivariate analysis, the variables associated independently with candidaemia due to C. albicans were: catheter (OR: 5.967; 95% CI: 1.614-22.057; P=.007) and prematurity (OR: 2.229; 95% CI: 1.141- 4.631; P=.020). CONCLUSIONS: The epidemiology of paediatric candidaemia varies between Spanish regions, but, globally, C. parapsilosis and C. albicans, are respectively, the first and second most frequently isolated species, and they show resistance rates to fluconazole of less than 5%.

Cellular human CLE/C14orf166 protein interacts with influenza virus polymerase and is required for viral replication.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including RNA polymerase II. We previously described the interaction of influenza virus polymerase subunit PA with human CLE/C14orf166 protein (hCLE), a positive modulator of this cellular RNA polymerase. Here, we show that hCLE also interacts with the influenza virus polymerase complex and colocalizes with viral ribonucleoproteins. Silencing of hCLE causes reduction of viral polymerase activity, viral RNA transcription and replication, virus titer, and viral particle production. Altogether, these findings indicate that the cellular transcription factor hCLE is an important protein for influenza virus replication.

CHD6 chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection.

The influenza virus establishes close functional and structural connections with the nucleus of the infected cell. Thus, viral ribonucleoproteins (RNPs) are closely bound to chromatin components and the main constituent of viral RNPs, the nucleoprotein (NP) protein, interacts with histone tails. Using a yeast two-hybrid screening, we previously found that the PA influenza virus polymerase subunit interacts with the CHD6 protein, a member of the CHD family of chromatin remodelers. Here we show that CHD6 also interacts with the viral polymerase complex and colocalizes with viral RNPs in the infected cells. To study the relationships between RNPs, chromatin and CHD6, we have analysed whether NP and CHD6 binds to peptides representing trimethylated lysines of histone 3 tails that mark transcriptionally active or inactive chromatin. Upon infection, NP binds to marks of repressed chromatin and, interestingly an important recruitment of CHD6 to these heterochromatin marks occurs in this situation. Silencing experiments indicate that CHD6 acts as a negative modulator of influenza virus replication. Hence, the CHD6 association with inactive chromatin could be part of a process where the influenza virus triggers modifications of chromatin-associated proteins that could contribute to the pathogenic events used by the virus to induce host cell shut-off.

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

AIMS: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. METHODS AND RESULTS: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. CONCLUSION: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.

Deletion of the Chd6 exon 12 affects motor coordination.

Members of the CHD protein family play key roles in gene regulation through ATP-dependent chromatin remodeling. This is facilitated by chromodomains that bind histone tails, and by the SWI2/SNF2-like ATPase/helicase domain that remodels chromatin by moving histones. Chd6 is ubiquitously expressed in both mouse and human, with the highest levels of expression in the brain. The Chd6 gene contains 37 exons, of which exons 12-19 encode the highly conserved ATPase domain. To determine the biological role of Chd6, we generated mouse lines with a deletion of exon 12. Chd6 without exon 12 is expressed at normal levels in mice, and Chd6 Exon 12 -/- mice are viable, fertile, and exhibit no obvious morphological or pathological phenotype. Chd6 Exon 12 -/- mice lack coordination as revealed by sensorimotor analysis. Further behavioral testing revealed that the coordination impairment was not due to muscle weakness or bradykinesia. Histological analysis of brain morphology revealed no differences between Chd6 Exon 12 -/- mice and wild-type (WT) controls. The location of CHD6 on human chromosome 20q12 is overlapped by the linkage map regions of several human ataxias, including autosomal recessive infantile cerebellar ataxia (SCAR6), a nonprogressive cerebrospinal ataxia. The genomic location, expression pattern, and ataxic phenotype of Chd6 Exon 12 -/- mice indicate that mutations within CHD6 may be responsible for one of these ataxias.

Attenuated strains of influenza A viruses do not induce degradation of RNA polymerase II.

We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.

Mutations of the segment-specific nucleotides at the 3' end of influenza virus NS segment control viral replication.

The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3' and 5' termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3' end. We have performed systematic point-mutations at the segment-specific nucleotides 15-18 of the 3'-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16-18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication.

Interferon- Stimulation Elicited by the Influenza Virus Is Regulated by the Histone Methylase Dot1L through the RIG-I-TRIM25 Signaling Axis.

Influenza virus infection increases the methylation of lysine 79 of histone 3 catalyzed by the Dot1L enzyme. The role of Dot1L against infections was highlighted by an increase of influenza A and vesicular stomatitis virus replication in Dot1L-inhibited cells mediated by a decreased antiviral response. Interferon-beta (IFN-ß) reporter assays indicate that Dot1L is involved in the control of retinoic acid-inducible geneI protein (RIG-I) signaling. Accordingly, Dot1L inhibition decreases the IFN-ß promoter stimulation and RIG-I- mitochondria-associated viral sensor (RIG-I-MAVS) association upon viral infection. Replication of an influenza A virus lacking NS1 (delNS1), incapable of counteracting the antiviral response, is not affected by Dot1L inhibition. Consequently, RIG-I-MAVS association and nuclear factor-B (NF-κ nuclear translocation, are not affected by the Dot1L inhibition in delNS1 infected cells. Restoration of NS1 expression in trans also reinstated Dot1L as a regulator of the RIG-I-dependent signaling in delNS1 infections. Interferon-inducible E3 ligase tripartite motif-containing protein 25 (TRIM25) expression increases in influenza virus infected cells, but Dot1L inhibition reduces both the TRIM25 expression and TRIM25 protein levels. TRIM25 overexpression reverses the defective innate response mediated by Dot1L inhibition elicited upon virus infection or by overexpression of RIG-I signaling intermediates. Thus, TRIM25 is a control point of the RIG-I recognition pathway controlled by Dot1L and may have a general role in RNA viruses recognized by the RIG-I sensor.

The DOT1L inhibitor Pinometostat decreases the host-response against infections: Considerations about its use in human therapy.

Patients with acute myeloid leukemia frequently present translocations of MLL gene. Rearrangements of MLL protein (MLL-r) in complexes that contain the histone methyltransferase DOT1L are common, which elicit abnormal methylation of lysine 79 of histone H3 at MLL target genes. Phase 1 clinical studies with pinometostat (EPZ-5676), an inhibitor of DOT1L activity, demonstrated the therapeutic potential for targeting DOT1L in MLL-r leukemia patients. We previously reported that down-regulation of DOT1L increases influenza and vesicular stomatitis virus replication and decreases the antiviral response. Here we show that DOT1L inhibition also reduces Sendai virus-induced innate response and its overexpression decreases influenza virus multiplication, reinforcing the notion of DOT1L controlling viral replication. Accordingly, genes involved in the host innate response against pathogens (RUBICON, TRIM25, BCL3) are deregulated in human lung epithelial cells treated with pinometostat. Concomitantly, deregulation of some of these genes together with that of the MicroRNA let-7B, may account for the beneficial effects of pinometostat treatment in patients with MLL-r involving DOT1L. These results support a possible increased vulnerability to infection in MLL-r leukemia patients undergoing pinometostat treatment. Close follow up of infection should be considered in pinometostat therapy to reduce some severe side effects during the treatment.