Negative Regulation of Interferon-β Gene Expression during Acute and Persistent Virus Infections

The production of type I interferons (IFNs) in response to viral infections is critical for antiviral immunity. However, IFN production is transient, and continued expression can lead to inflammatory or autoimmune diseases. Thus, understanding the mechanisms underlying the negative regulation of IFN expression could lead to the development of novel therapeutic approaches to the treatment of these diseases. We report that the transcription factor IRF3 plays a central role in the negative regulation of interferon-β (IFNβ) expression during both acute and persistent (chronic) virus infections. We show that the degradation of IRF3 during acute infections, rather than the activation of transcriptional repressors, leads to the down regulation of IFNβ expression. We also show that the block to IFNβ expression in mouse embryonic fibroblasts that are persistently infected with Sendai virus (SeV) correlates with the absence of transcriptionally active IRF3. Remarkably, ongoing protein synthesis and viral replication are required to maintain repression of the IFNβ gene in persistently infected cells, as the gene can be activated by the protein synthesis inhibitor cycloheximide, or by the antiviral drug ribavirin. Finally, we show that the SeV V protein inhibits IRF3 activity in persistently infected cells. Thus, in conjunction with the known interference with STAT1 by the SeV C protein, both IFN activation and its signaling pathways are blocked in persistently infected cells. We conclude that the transcription factor IRF3 is targeted for turnover and inactivation through distinct mechanisms from both the host cells and virus, leading to the inhibition of IFNβ gene expression during acute and persistent viral infections. These observations show that IRF3 plays a critical role, not only in the activation of the IFNβ gene, but also in the controlling the duration of its expression. (284 words

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Group A Streptococcus Prevents Mast Cell Degranulation to Promote Extracellular Trap Formation

The resurgence of Group A Streptococcus (GAS) infections in the past two decades has been a rising major public health concern. Due to a large number of GAS infections occurring in the skin, mast cells (MCs), innate immune cells known to localize to the dermis, could play an important role in controlling infection. MCs can exert their antimicrobial activities either early during infection, by degranulation and release of antimicrobial proteases and the cathelicidin-derived antimicrobial peptide LL-37, or by forming antibacterial MC extracellular traps (MCETs) in later stages of infection. We demonstrate that MCs do not directly degranulate in response to GAS, reducing their ability to control bacterial growth in early stages of infection. However, MC granule components are highly cytotoxic to GAS due to the pore-forming activity of LL-37, while MC granule proteases do not significantly affect GAS viability. We therefore confirmed the importance of MCETs by demonstrating their capacity to reduce GAS survival. The data therefore suggests that LL-37 from MC granules become embedded in MCETs, and are the primary effector molecule by which MCs control GAS infection. Our work underscores the importance of a non-traditional immune effector cell, utilizing a non-conventional mechanism, in the defense against an important human pathogen

Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.]

Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection

<div><p>Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in <em>RNF5^−/−</em> MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in <em>RNF5^−/−</em> using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in <em>Caenorhabditis elegans</em> increases the level of LGG-1/LC3::GFP puncta. <em>RNF5^−/−</em> mice are more resistant to group A <em>Streptococcus</em> infection, associated with increased autophagosomes and more efficient bacterial clearance by <em>RNF5^−/−</em> macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.</p> </div

RNF-5 regulates autophagy in <i>C. elegans</i>.

<p>(A–C) Representative images of seam cells from L3 larvae expressing the GFP::LGG-1 transgene, and the average number of GFP::LGG-1 puncta in seam cells. Three to four independent experiments were performed for each condition. Error bars are ± SEM. P value was calculated using an unpaired two-tailed t-test. (A) <i>rnf-5(tm794)</i> larvae had an average of 5.53±0.49 puncta/cell compared to 2.11±0.30 puncta/cell in WT (36 cells from 9 <i>rnf-5(tm794)</i> larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) <i>rnf-5(RNAi)</i>-treated worms had an average of 4.42±0.47 puncta/cell compared to 1.83±0.31 puncta/cell in control animals (77 cells from 15 <i>rnf-5(RNAi)</i> larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: <i>hsp-16p</i>::<i>rnf-5</i> larvae had an average of 1.71±0.24 puncta/cell compared to 3.75±0.34 puncta/cell in the non-transgenic population (69 cells from 18 <i>hsp-16p</i>::<i>rnf-5</i> larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).</p

RNF5 negatively regulates autophagy.

<p>(A) Immunoblot analysis of LC3 and p62. RNF5 WT and KO MEFs were maintained in HBSS for 1 to 4 h before cells were lysed, and proteins were resolved and analyzed by immunoblotting with the indicated antibodies. (B) Scrambled and shRNF5-transduced PC3 cells were maintained in normal medium, serum-starved overnight, or treated with tunicamycin (TM, 5 µg/ml, 8 h). (C) Amount of endogenous LC3 puncta is affected by RNF5. RNF5 WT and KO MEFs grown in normal medium, starved with HBSS (2 h), or treated with DTT (5 mM, 8 h) were fixed and immunostained with anti-LC3 antibody. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003007#s2" target="_blank">Results</a> show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.</p

RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.

<p>(A) Membrane-bound RNF5 interacts with ATG4B. RNF5 WT, C-terminal TM-deleted mutant (dCT), and RING domain mutant (RM) constructs were co-transfected with Flag-ATG4B. The cell lysates were immunoprecipitated with anti-Flag antibodies conjugated to beads, then immunoblotted with the indicated antibodies. (B) Dynamic interaction of ATG4B and RNF5 during starvation-induced autophagy. The interaction between endogenous ATG4B and RNF5 expressed in HeLa cells was monitored at the indicated time points prior to and following HBSS-induced starvation. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (C) In vivo ATG4B ubiquitination by RNF5. HeLa cells were co-transfected with Flag-ATG4B, HA-ubiquitin (Ub), and WT or RM RNF5 plasmids. After 24 h, cells were treated with MG132 (10 µM) for 4 h and lysed with buffer containing 1% SDS. The lysates were immunoprecipitated with anti-Flag antibody in the presence of 0.1% SDS, followed by immunoblotting with anti-HA antibodies. (D) ATG4B in vitro ubiquitination by RNF5. Purified His6-tagged ATG4B was bound to nickel beads and incubated for 1 h at 37°C with RNF5 in the presence of the indicated in vitro ubiquitination reagents. The bead were washed three times with PBS containing 0.1% SDS and 0.2% Triton X-100 and then immunoblotted with antibodies to ATG4B and Ub. (E) RNF5 reduces ATG4B stability. Half-life of ATG4B in RNF5 WT and KO MEFs under normal growth conditions or after HBSS treatment was determined by addition of cycloheximide (CHX) (40 µg/ml) for the indicated times. Cell extracts were subjected to immunoblot analysis using anti-ATG4B and anti-tubulin antibodies. Quantitation of ATG4B levels based on band intensity was measured using the LICOR system, and is shown as the mean of duplicate experiments.</p