A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection

<div><p>RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.</p></div

Overexpression of PUM1 and PUM2 results in enhanced NDV-induced <i>IFNB</i> promoter activity.

<p>(A) Schematic representation of PUM1 and PUM2. PUM-HD shows high sequence similarity between PUM1 and PUM2. Positions of histidine (H) residues critical for NRE recognition are indicated. (B–D) L929 cells were transfected with the indicated reporter gene, p-125Luc (B), p-55C1BLuc (C) or p-55A2Luc (D), and pRL-tk, together with the expression vector for PUM1 or PUM2. The cells were stimulated by NDV infection for 9 h and subjected to a dual-luciferase assay. (E) L929 cells were transfected with an expression vector for PUM1 or PUM2. The cells were infected with NDV for 24 h, and then NDV RNA levels were determined by quantitative RT-PCR. (F) L929 cells were transfected with p-125Luc and pRL-tk, together with the expression vector for wt and histidine mutants of PUM1 or PUM2 as indicated. The cells were stimulated by NDV infection and subjected to a dual-luciferase assay. Data are from one representative of at least two independent experiments; means and S.D. of duplicate experiments are shown (*p<0.05).</p

<i>In vitro</i> binding assay of dsRNA and LGP2.

<p>(A and B) Recombinant LGP2 proteins (0.125 µg) were mixed with ³²P-labeled dsRNA in the presence of a control mouse IgG or anti-Flag antibody (0.1, 0.2 or 0.4 µg) (A) or in the presence or absence of Pumilio proteins (0.1, 0.3 or 0.5 µg) (B). The mixture were separated by acrylamide gel and the radioactivity was analyzed. (C) LGP2 dsRNA binding affinities in the absence (filled circles) or presence of PUM1 (open square) or PUM2 (filled triangle) were analyzed and the Kd values were determined.</p

Hypothetical model for regulation of LGP2 by PUM1 and PUM2 in avSG.

<p>N-terminal domain of PUM1 and PUM2 possess intrinsic affinity to LGP2. This interaction confers higher binding affinity of LGP2 to viral dsRNA. Conformational change of LGP2 is one of the explanations for the increased affinity. Viral infection such as NDV induces avSGs and accumulation of viral dsRNA, LGP2, PUM1, PUM2 and other avSG markers into avSGs. Within avSG, dsRNA interacts with LGP2/PUM complex, producing LGP2/dsRNA complex and Pumilio proteins are released from the complex. Then, LGP2 triggers signals presumably in cooperation with RIG-I or MDA5. X: potential interacting partner of C-terminal domain of PUM1 and PUM2 determining their avSG localization.</p

Knockdown of PUM1 and PUM2 downregulates NDV-induced gene activation.

<p>(A–C) HEK293T cells were transfected with control siRNA (siN.C.) or siRNA targeting human PUM1 or PUM2 for 48 h. Knockdown efficiency was confirmed by immunoblotting with anti-PUM1, anti-PUM2 and anti-β-actin antibodies (A). The cells were infected with NDV for 9 h, and <i>IFNB</i> (B) and <i>CXCL10</i> (C) mRNA levels were determined by quantitative RT-PCR. (D and E) HEK293T cells were transfected with control siRNA or siRNA targeting PUM1 or PUM2 for 48 h. The cells were infected with NDV for 24 h. The culture media were collected and subjected to IFN-β ELISA (D). Total cellular RNA was extracted and subjected to qRT-PCR for NDV RNA (E). Data are from one representative of at least two independent experiments; means and S.D. of duplicate experiments are shown (*p<0.05, **p<0.01).</p

Cellular localization of PUM1, PUM2 and LGP2.

<p>(A–C) HeLa cells were mock-treated or infected with NDV for 9 h, fixed and stained with the indicated antibodies. Nuclei were stained with DAPI. (D) HEK293T cells were transfected with the expression vector for Flag-PUM1dN or Flag-PUM2dN for 48 h and mock treated or infected with NDV for 9 h. The cells were stained with anti-Flag or anti-TIAR.</p

Physical association of PUM1 and PUM2 with LGP2 and involvement of N- and C-terminal domains of PUM1 and PUM2 in IFN induction.

<p>(A) HEK293T cells were transfected with expression vector HA-tagged RIG-I, MDA5, LGP2 or IPS-1, together with Flag-tagged PUM1 or PUM2. For TRIM25, HEK293T cells were transfected with Flag-tagged PUM1 or PUM2, together with Myc-tagged TRIM25. The cell lysates were subjected to anti-Flag or anti-c-Myc immunoprecipitation (IP), followed by Western blotting. Western blotting result of total lysate is shown as a reference (Input, 5%). (B) L929 cells were transfected with control shRNA construct (pU6i-control) or shRNA for LGP2 (pU6i-LGP2#1 and #2) and expression vectors for PUM1 or PUM2 and p-125Luc reporter and pRLtk as indicated. The cells were stimulated by infection with NDV for 9 h and subjected to the dual luciferase assay. (C) Schematic representation of PUM1 or PUM2 deletion mutants used for IP experiments. (D) HEK293T cells were transfected with expression vectors for the indicated proteins and for HA-tagged LGP2. The cell lysates were subjected to IP with anti-Flag, followed by immunoblotting with anti-HA. Immunoblotting result of total lysate is shown as a reference (Input, 5%). (E) L929 cells were transfected with expression vectors for the wild type or mutant of PUM1 or PUM2 and p-125Luc reporter and pRL-tk as indicated. Cells were stimulated by infection with NDV for 9 h and subjected to the dual luciferase assay. Data are from one representative of at least two independent experiments (means and s.d. of duplicate experiments.)</p

Antiviral Activity of Phenolic Derivatives in Pyroligneous Acid from Hardwood, Softwood, and Bamboo

Pyroligneous acids (PA) from hardwood, softwood, and bamboo significantly disinfected encephalomyocarditis virus (EMCV). Twenty-five kinds of phenolic derivatives in the PAs were identified and quantified. The total amounts of phenolic compounds in bamboo PA is higher than those in the PAs from softwood and hardwood. Phenol, 2-methoxyphenol, 2-methoxy-4-methylphenol, and 2-methoxy-4-ethylphenol are the most abundant compounds in the PAs examined. The activities of all the phenolic compounds against the encephalomyocarditis virus were assessed. The number of phenolic hydroxyl groups significantly affects the antiviral activity, and catechol and its derivatives exhibit higher viral inhibition effects than other phenolic derivatives. In addition, substituents affect the antiviral activity of the compounds. Phenolic compounds with a methyl group show higher activities than with a methoxyl group (e.g., 2-methylphenol > 2-methoxyphenol). Moreover, the relative position of functional groups also plays a key role in the viral inhibition activity (e.g., 2,6-dimethoxyphenol > 3,4-dimethoxyphenol). Thus, PAs contain phenol derivatives with considerable structural diversity and viral inhibition activities, providing a new strategy for virus-inactivation treatment through the optimization of PA-derived phenol structures

Distinct genetic clades of enterovirus D68 detected in 2010, 2013, and 2015 in Osaka City, Japan

<div><p>The first upsurge of enterovirus D68 (EV-D68), a causative agent of acute respiratory infections (ARIs), in Japan was reported in Osaka City in 2010. In this study, which began in 2010, we surveyed EV-D68 in children with ARIs and analyzed sequences of EV-D68 strains detected. Real-time PCR of 19 respiratory viruses or subtypes of viruses, including enterovirus, was performed on 2,215 specimens from ARI patients (<10 years of age) collected between November 2010 and December 2015 in Osaka City, Japan. EV-D68 was identified in 18 enterovirus-positive specimens (<i>n</i> = 4 in 2013, <i>n</i> = 1 in 2014, and <i>n</i> = 13 in 2015) by analysis of viral protein 1 (VP1) or VP4 sequences, followed by a BLAST search for similar sequences. All EV-D68 strains were detected between June and October (summer to autumn), except for one strain detected in 2014. A phylogenetic analysis of available VP1 sequences revealed that the Osaka strains detected in 2010, 2013, and 2015 belonged to distinct clusters (Clades C, A, and B [Subclade B3], respectively). Comparison of the 5′ untranslated regions of these viruses showed that Osaka strains in Clades A, B (Subclade B3), and C commonly had deletions at nucleotide positions 681–703 corresponding to the prototype Fermon strain. Clades B and C had deletions from nucleotide positions 713–724. Since the EV-D68 epidemic in 2010, EV-D68 re-emerged in Osaka City, Japan, in 2013 and 2015. Results of this study indicate that distinct clades of EV-D68 contributed to re-emergences of this virus in 2010, 2013, and 2015 in this limited region.</p></div

Alignment of nucleotide sequences of the 5′ UTR of strains in the three major genetic clades of EV-D68.

<p>Nucleotide sequences (nucleotide positions 501–1,000, corresponding to those of the Fermon strain) were aligned in MEGA 7.0. A partial sequence (nucleotide positions 671–740 of Fermon strain) is shown. Hyphens denote deleted nucleotides.</p