Epigenetic control of influenza virus: role of H3K79 methylation in interferon-induced antiviral response

Influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. Using an unbiased search, we analyzed the epigenetic changes at DNA methylation and post-translational histone modification levels induced by the infection. DNA methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. A particular increase in H3K79 methylation was observed and the use of an inhibitor of the specific H3K79 methylase, Dot1L enzyme, or its silencing, increased influenza virus replication. The antiviral response was reduced in conditions of Dot1L downregulation, since decreased nuclear translocation of NF-kB complex, and IFN-β, Mx1 and ISG56 expression was detected. The data suggested a control of antiviral signaling by methylation of H3K79 and consequently, influenza virus replication was unaffected in IFN pathway-compromised, Dot1L-inhibited cells. H3K79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. Epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens

The Splicing Factor Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection

Interacciones del virus de la gripe con la célula hospedadora: identificación de dianas celulares con potencial para la inhibición del virus

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-07-2014The influenza A viruses are members of the Orthomyxoviridae family and cause yearly epidemics and occasional more severe pandemics of respiratory infections. Although influenza in humans is usually resolved without complications, the infection can be more severe in the young infants, in the elderly and in persons with previous medical conditions. Thus, influenza leads to 250.000-500.000 deaths per year and these numbers can be much larger in a pandemic. The genome of influenza A viruses is fragmented into 8 single-stranded RNA molecules of negative polarity that together with the heterotrimeric polymerase complex (PA, PB1 and PB2) and the nucleoprotein (NP), form ribonucleoprotein complexes (RNPs). These RNPs are responsible for the replication and transcription of the virus genes in the nucleus of the infected cells. Although at present we have effective influenza antivirals available targeting virus dissemination, the appearance of resistant mutant viruses poses a serious limitation for their widespread use. Antivirals drugs that target cellular proteins may play major roles in combating virus resistance. In the present work we propose two different strategies to identify cellular proteins with potential antiviral activity. The first strategy is based in the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. Several influenza polymerase-associated proteins were identified by MALDI-MS. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2-5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4-5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection. The second strategy is based in the screen for new anti-influenza inhibitors targeted to viral RNA replication or transcription in a recombinant virus replicon system. We proposed to use collections of compounds that have been previously studied for the therapy of human pathologies and hence have known toxicology and pharmacological profiles. This approach could allow the rapid progress to the market of new effective influenza antivirals. The compounds that showed activity in the basic screen were tested for their anti-influenza activity in cultured infected cells. Two compounds inhibited the influenza virus strains used in cell cultures from different origins: Ribavirin, a previously described anti-influenza compound, and Montelukast sodium. Subtoxic concentrations of Montelukast sodium inhibited the viral proteins synthesis. The identification of the target of this compound could reveal new cellular pathways important for influenza virus infection

Epigenetic control of influenza virus: role of H3K79 methylation in interferon-induced antiviral response

Influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. Using an unbiased search, we analyzed the epigenetic changes at DNA methylation and post-translational histone modification levels induced by the infection. DNA methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. A particular increase in H3K79 methylation was observed and the use of an inhibitor of the specific H3K79 methylase, Dot1L enzyme, or its silencing, increased influenza virus replication. The antiviral response was reduced in conditions of Dot1L downregulation, since decreased nuclear translocation of NF-kB complex, and IFN-β, Mx1 and ISG56 expression was detected. The data suggested a control of antiviral signaling by methylation of H3K79 and consequently, influenza virus replication was unaffected in IFN pathway-compromised, Dot1L-inhibited cells. H3K79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. Epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens

Epigenetic control of influenza virus: role of H3K79 methylation in interferon-induced antiviral response

Influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. Using an unbiased search, we analyzed the epigenetic changes at DNA methylation and post-translational histone modification levels induced by the infection. DNA methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. A particular increase in H3K79 methylation was observed and the use of an inhibitor of the specific H3K79 methylase, Dot1L enzyme, or its silencing, increased influenza virus replication. The antiviral response was reduced in conditions of Dot1L downregulation, since decreased nuclear translocation of NF-kB complex, and IFN-β, Mx1 and ISG56 expression was detected. The data suggested a control of antiviral signaling by methylation of H3K79 and consequently, influenza virus replication was unaffected in IFN pathway-compromised, Dot1L-inhibited cells. H3K79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. Epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens

Sudan Ebolavirus VP35-NP Crystal Structure Reveals a Potential Target for Pan-Filovirus Treatment

Outbreaks of the filoviruses can be unpredictable in timing, location, and identity of the causative virus, with each of Ebola virus, Sudan virus, Bundibugyo virus, and Marburg virus reemerging in the last several years to cause human disease with 30 to 90% lethality. The 2014–2016 outbreak in particular, with nearly 30,000 patients, highlighted the ability of these viruses to emerge unexpectedly and spread rapidly. Two ebolavirus outbreaks have emerged this year, yet we still lack FDA-approved drugs with pan-filovirus activity to treat existing and emergent ebolaviruses. For all filoviruses, the interaction between the nucleoprotein and the phosphoprotein is essential for the virus life cycle and is a potential target for therapeutic intervention. In this report, we describe the crystal structure of the SUDV nucleoprotein with the interacting domain of the viral phosphoprotein, and we identify residues critical for high-affinity interaction and for control of the oligomeric state of the nucleoprotein. Structural comparison of this heterodimer with other members of the filovirus family allowed us to find conserved and essential atomic features that will facilitate understanding of the virus life cycle and the rational design of antivirals.The filoviruses are etiological agents of life-threatening hemorrhagic fever with high mortality rate and risk of potential outbreak. Among members of this family, the Ebola (EBOV), Sudan (SUDV), and Marburg (MARV) viruses are considered the most pathogenic for humans. The ebolavirus nucleoprotein (NP) is the most abundant protein in infected cells and is essential for viral transcription and replication; thus, it represents an attractive target for therapeutic intervention. Here, we present the structure of SUDV NP in complex with the amino-terminal portion of the phosphoprotein VP35 at 2.3 Å. This structure captures VP35 chaperoning SUDV NP in a monomeric and RNA-free state. This transient state has been proposed to be key to maintaining a pool of monomeric and RNA-free NPs prior to NP-NP polymerization and encapsidation of the viral RNA genome. This structure also reveals a newly visualized interaction between NP and VP35, a well-defined beta sheet that is not present in previous structures. Affinity binding assays demonstrate that this beta sheet is essential for maintaining the high-affinity interaction between VP35 and a hydrophobic pocket on SUDV NP, and electron microscopy indicates the importance of this binding interaction to the oligomeric state and assembly of NP in human cells. Complementary structure-directed mutagenesis identifies critical residues conserved across the filovirus family that could be targeted by broadly effective antivirals

hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/ C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; BFU2011-26175 and BFU2014-57797-R) and the Ciber de Enfermedades Respiratorias (ISCIII).Peer reviewe

Human coronavirus OC43-elicited CD4+ T cells protect against SARS-CoV-2 in HLA transgenic mice

Abstract SARS-CoV-2-reactive T cells are detected in some healthy unexposed individuals. Human studies indicate these T cells could be elicited by the common cold coronavirus OC43. To directly test this assumption and define the role of OC43-elicited T cells that are cross-reactive with SARS-CoV-2, we develop a model of sequential infections with OC43 followed by SARS-CoV-2 in HLA-B*0702 and HLA-DRB1*0101 Ifnar1 −/− transgenic mice. We find that OC43 infection can elicit polyfunctional CD8+ and CD4+ effector T cells that cross-react with SARS-CoV-2 peptides. Furthermore, pre-exposure to OC43 reduces subsequent SARS-CoV-2 infection and disease in the lung for a short-term in HLA-DRB1*0101 Ifnar1 −/− transgenic mice, and a longer-term in HLA-B*0702 Ifnar1 −/− transgenic mice. Depletion of CD4+ T cells in HLA-DRB1*0101 Ifnar1 −/− transgenic mice with prior OC43 exposure results in increased viral burden in the lung but no change in virus-induced lung damage following infection with SARS-CoV-2 (versus CD4+ T cell-sufficient mice), demonstrating that the OC43-elicited SARS-CoV-2 cross-reactive T cell-mediated cross-protection against SARS-CoV-2 is partially dependent on CD4+ T cells. These findings contribute to our understanding of the origin of pre-existing SARS-CoV-2-reactive T cells and their effects on SARS-CoV-2 clinical outcomes, and also carry implications for development of broadly protective betacoronavirus vaccines

A Zika virus mutation enhances transmission potential and confers escape from protective dengue virus immunity.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest