SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability.

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

The administration of oseltamivir results in reduced effector and memory CD8+ T cell responses to influenza and affects protective immunity.

The clinical benefits of oseltamivir (Tamiflu) are well established, but the effects of antiviral treatment on the immune response are poorly understood. By use of flow cytometric analyses and the mouse model, we thoroughly investigated the impact of such a treatment on the immune response and the generation of protective immunity to influenza. We demonstrated that influenza-specific CD8(+) effector T cell recruitment was reduced up to 81% in the lungs of mice treated with oseltamivir (5 or 50 mg/kg twice daily; EC50 49 nM in vitro) compared to saline controls, but cell generation was unaffected in draining lymph nodes. Importantly, we showed that oseltamivir administration significantly decreased the pools of tissue-resident and circulating effector memory (93.7%) and central memory CD8(+) T cells (45%) compared to saline controls. During heterologous secondary infection, a decreased memory CD8(+) T cell pool combined with reduced generation of secondary influenza-specific effectors in the lymph nodes resulted in 10-fold decreased CD8(+) T cell recall responses, which increased mouse morbidity and delayed viral clearance. Furthermore, antiviral administration led to a significant 5.7-fold decreased production of functional anti-influenza antibodies. Thus, our study demonstrates that antiviral treatment affects the development of the adaptive immune response and protective immunity against influenza.

Instability of trinucleotidic repeats during chromatin remodeling in spermatids.

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

The prostanoid 15-deoxy-Δ12,14-prostaglandin-j2 reduces lung inflammation and protects mice against lethal influenza infection.

BACKGROUND: Growing evidence indicates that influenza pathogenicity relates to altered immune responses and hypercytokinemia. Therefore, dampening the excessive inflammatory response induced after infection might reduce influenza morbidity and mortality. METHODS: Considering this, we investigated the effect of the anti-inflammatory molecule 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) in a mouse model of lethal influenza infection. RESULTS: Administration of 15d-PGJ(2) on day 1 after infection, but not on day 0, protected 79% of mice against lethal influenza infection. In addition, this treatment considerably reduced the morbidity associated with severe influenza infection. Our results also showed that treatment with 15d-PGJ(2) decreased influenza-induced lung inflammation, as shown by the diminished gene expression of several proinflammatory cytokines and chemokines. Unexpectedly, 15d-PGJ(2) also markedly reduced the viral load in the lungs of infected mice. This could be attributed to maintained type I interferon gene expression levels after treatment. Interestingly, pretreatment of mice with a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist before 15d-PGJ(2) administration completely abrogated its protective effect against influenza infection. CONCLUSIONS: Our results demonstrate for the first time that treatment of mice with 15d-PGJ(2) reduces influenza morbidity and mortality through activation of the PPARγ pathway. PPARγ agonists could thus represent a potential therapeutic avenue for influenza infections.

Infection with a Mouse-Adapted Strain of the 2009 Pandemic Virus Causes a Highly Severe Disease Associated with an Impaired T Cell Response.

Despite a relatively low fatality rate, the 2009 H1N1 pandemic virus differed from other seasonal viruses in that it caused mortality and severe pneumonia in the young and middle-aged population (18-59 years old). The mechanisms underlying this increased disease severity are still poorly understood. In this study, a human isolate of the 2009 H1N1 pandemic virus was adapted to the mouse (MAp2009). The pathogenicity of the MAp2009 virus and the host immune responses were evaluated in the mouse model and compared to the laboratory H1N1 strain A/Puerto Rico/8/1934 (PR8). The MAp2009 virus reached consistently higher titers in the lungs over 14 days compared to the PR8 virus, and caused severe disease associated with high morbidity and 85% mortality rate, contrasting with the 0% death rate in the PR8 group. During the early phase of infection, both viruses induced similar pathology in the lungs. However, MAp2009-induced lung inflammation was sustained until the end of the study (day 14), while there was no sign of inflammation in the PR8-infected group by day 10. Furthermore, at day 3 post-infection, MAp2009 induced up to 10- to 40-fold more cytokine and chemokine gene expression, respectively. More importantly, the numbers of CD4+ T cells and virus-specific CD8+ T cells were significantly lower in the lungs of MAp2009-infected mice compared to PR8-infected mice. Interestingly, there was no difference in the number of dendritic cells in the lung and in the draining lymph node. Moreover, mice infected with PR8 or MAp2009 had similar numbers of CCR5 and CXCR3-expressing T cells, suggesting that the impaired T cell response was not due to a lack of chemokine responsiveness or priming of T cells. This study demonstrates that a mouse-adapted virus from an isolate of the 2009 pandemic virus interferes with the adaptive immune response leading to a more severe disease.

Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

Inhibition of influenza virus replication by targeting broad host cell pathways.

Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs) and calcium modulators (CMs), which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA) revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.

Initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract.

Factors from the virus and the host contribute to influenza virus pathogenicity and to the development of immunity. This study thoroughly examined the effects of an initial infectious dose of virus and unveiled new findings concerning the antiviral and inflammatory responses, innate and adaptive immunity, memory responses, and protection against secondary heterologous infection. Our results demonstrated that the initial infectious dose significantly affects the gene expression of antiviral (IFN-ß) and inflammatory (TNF-α, IL-6, IL-1ß) cytokines and of enzymes involved in nitrosative/oxidative stress (iNOS, HO-1, NQO1) early in the response to influenza. This response correlated with significantly increased recruitment of innate immune cells into the lungs of infected mice. We showed that this response also alters the subsequent accumulation of activated IFN-γ(+) CD44(hi) CD62L(lo) influenza-specific CD8(+) T cells into the lungs of infected mice through increased T cell-recruiting chemokine gene expression (CCL3, CCL4, CCL5, CXCL10). Furthermore, we demonstrated that the initial infectious dose determines the generation and the distribution of memory CD8(+) T cell subsets without affecting trafficking mechanisms. This impacted on immune protection against heterologous infection. Lastly, we showed that the effects on innate and adaptive immunity were not dependent on influenza strain or on the genetic background of the host. Collectively, our data show for the first time and in detail that the initial infectious dose of influenza determines the development of several aspects of antiviral immunity. This study provides new insights on virus-host interaction in the generation of the global immune response to influenza.