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Pathogenesis and Immunity
Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing
Mary J. Manuse, Griffith D. Parks
Mary J. Manuse
Department of Microbiology and Immunology, Wake Forest University, School of Medicine, Winston-Salem, North Carolina 27157-1064
Griffith D. Parks
For correspondence: gparks@wfubmc.edu
The parainfluenza virus simian virus 5 (SV5) is a poor inducer of innate immune responses. In contrast, the naturally occurring SV5 variant Wake Forest parainfluenza virus (WF-PIV) activates the synthesis of proinflammatory cytokines and beta interferon (IFN-β). Comparison of SV5 and WF-PIV genome sequences revealed nine nucleotide differences within the viral genomic promoter, including two substitutions (U5C and A14G) in the most highly conserved 3′-end promoter element. To test the consequences of these promoter variations, a recombinant SV5 mutant [Le-(U5C, A14G)] was engineered to harbor the two WF-PIV genomic promoter substitutions in an otherwise wild-type (WT) SV5 background. Human lung epithelial cells infected with the Le-(U5C, A14G) mutant had higher rates of viral protein synthesis and levels of mRNA than cells infected with WT SV5, but levels of genomic RNA were not changed. Unlike WT SV5, the Le-(U5C, A14G) mutant was a potent inducer of interleukin-6 and IFN-β synthesis, despite expressing a functional V protein antagonist. Cytokine responses to Le-(U5C, A14G) infection were reduced either by small interfering RNA-mediated knockdown of retinoic acid-inducible gene I (RIG-I) or after infection of cells that were engineered to express the reovirus sigma3 double-stranded RNA-binding protein. Le-(U5C, A14G) induced cytopathic effects not seen with WT SV5, and the extent of cell killing correlated with elevated levels of viral F protein and cell-cell fusion. Our results support a model whereby the SV5 promoter has evolved to function at an attenuated level in order to limit (i) synthesis of aberrant RNAs which induce RIG-I-mediated responses and (ii) overproduction of mRNA for potentially toxic gene products, such as the F protein. Control of genomic promoter activity may be particularly important for viruses such as SV5, that express a V protein targeting mda-5 but do not encode antagonists such as the paramyxovirus C proteins, that specifically target RIG-I.
Innate host cell responses to viral infection are important factors that play major roles in viral pathogenesis, adaptive immunity, and tropism for specific tissues or cells (2). These antiviral responses include the stimulation of type I interferon (IFN) pathways and the secretion of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 (21, 41). Paramyxoviruses, such as simian virus 5 (SV5), can prevent activation of host antiviral responses by directly inhibiting the pathways that lead to activation (4) or by attenuating the expression of viral products that are the activators of a host cell response (9). Here we provide evidence for the role of the viral genomic promoter in enabling SV5 to avoid activation of innate responses.
Members of the paramyxovirus family of negative-strand RNA viruses employ a diverse range of mechanisms to circumvent host cell antiviral responses (reviewed in references 7 and 15). Many of these mechanisms for counteracting cytokine responses have been attributed to products of the paramyxovirus P/V (or P/V/C) gene, which, depending on the particular virus, encodes the phosphoprotein P subunit of the RNA-dependent RNA polymerase, the accessory V protein, and in some cases the family of C proteins (8, 26, 14, 33). For SV5, accurate transcription of the P/V gene results in an mRNA coding for the V protein. The P mRNA is identical to the V mRNA except for the addition of two nontemplated G residues that are inserted by the viral polymerase at a precise location in the P/V transcript (44). The P and V proteins have unique C-terminal regions, with the V protein encoding a highly conserved cysteine-rich domain that is required for many V-associated functions (8, 17, 38).
A major function of the SV5 V protein is the inhibition of IFN signaling, which occurs through V-mediated targeting of signal transducer and activator of transcription 1 (STAT1) for ubiquitylation and degradation (1, 8, 45). The SV5 V protein also blocks activation of the IFN-β promoter during virus infection (17) or following transfection of double-stranded RNA (dsRNA) (4, 38). The paramyxovirus V protein inhibits IFN-β induction by targeting the IFN-inducible RNA helicase encoded by the melanoma differentiation-associated gene 5 (mda-5) (4, 5). Inhibition of mda-5 activation requires only the V protein cysteine-rich C-terminal domain (4, 38), which prevents mda-5 from binding dsRNA and self-association (5). In contrast, the alternative RNA helicase retinoic acid-inducible gene I (RIG-I) does not appear to be inhibited by V protein (4, 5). V protein also appears to act as a decoy substrate for kinases that activate IFN regulatory factor 3 (IRF-3) (29) and IRF-7 (36). Similarly, the SV5 V protein is reported to block IL-6 induction (27), although virus-infected cells still respond to tumor necrosis factor alpha-mediated induction of IL-6 (50).
In addition to the V protein, some paramyxoviruses, such as Sendai virus (SeV) and measles virus, encode a group of C proteins that are expressed from the P/V/C gene (26). The SeV C protein can prevent IFN-β induction (42) by a mechanism that is thought to involve the inhibition of RIG-I activation (42). Thus, paramyxoviruses that express both V and C proteins have the capacity to block both mda-5 via the V protein and RIG-I via the C proteins. Rubulaviruses, such as SV5 and mumps virus, express a V protein but do not encode C proteins (26). This raises the important question of how SV5 can establish a robust, highly productive infection of epithelial cells without activation of RIG-I.
Wild-type (WT) SV5 is a poor inducer of host cell responses, including the activation of proinflammatory cytokines and IFN-β (6, 17, 46). In contrast, we have shown previously that the naturally occurring SV5 variant Wake Forest isolate of parainfluenza virus (WF-PIV) has a delay in viral RNA synthesis but is a potent inducer of IL-8 and IFN-β responses (50). WF-PIV is closely related to the CPI strain of SV5 and was isolated in our lab from persistently infected Vero cells (50). Sequence analysis has identified many nucleotide differences between WF-PIV and WT SV5 (50). Exchanging the P/V gene from WF-PIV or a second variant, CPI-, into an otherwise WT SV5 background results in a chimeric virus that overexpresses viral gene products and activates cytokine secretion and apoptosis (10, 46, 50). These results illustrate that chimeras can have phenotypes that are very different from those of the two parental viruses (10) and suggest that the trans-acting paramyxovirus gene products function in concert to coordinate viral gene expression and limit host cell responses. Here we show that the sequence of the WF-PIV genomic promoter differs substantially from that of WT SV5 in a key regulatory domain, and we analyze the consequences of variability in these cis-acting sequences for gene expression and antiviral responses.
The paramyxovirus genome contains a promoter at its 3′ end that directs both mRNA transcription and genome replication (26) and is transcribed into a short noncoding leader RNA. For many paramyxoviruses, such as SV5, SeV, and human parainfluenza virus type 3, the genomic promoter contains two 18-base promoter elements (PrE) that are essential for viral RNA synthesis (19, 43, 31, 32). PrE-I is located at the ultimate 3′ end of the genome and is separated from a second internal PrE, PrE-II, by a sequence-independent spacer region (Fig. 1). The WF-PIV genomic promoter differs from that of WT SV5 at nine positions, but unexpectedly two of the WF-PIV changes are in the highly conserved PrE-I. We introduced these two nucleotide changes into the SV5 genomic promoter and created a mutant virus which overexpressed viral mRNA and protein. Despite expressing a functional V protein, this promoter mutant virus unexpectedly also activated host cell cytokine responses through pathways that were dependent on RIG-I and dsRNA. Our results support a model whereby the SV5 genomic promoter plays a role in limiting production of aberrant viral RNA that can trigger RIG-I-mediated antiviral pathways. Control of genomic promoter activity may be particularly important for viruses such as SV5, that express a V protein targeting mda-5 but do not encode antagonists such as the C proteins, that specifically target RIG-I (42).
Schematic diagram of SV5 genomic promoter and viruses used in this study. (A) Genomic promoter. The 3′-to-5′ sequence of the 90-base SV5 genomic promoter is shown, with the positions of nucleotides that differ between WT SV5 (top) and the WF-PIV variant of SV5 (bottom). The three hexamers that compose PrE-I and PrE-II are denoted by underlining, and the start site for transcription of the NP gene is shown by an arrow. (B) The genome structure of SV5 is shown, with the addition of the GFP gene between HN and L, as described previously (16). The sequences at positions 5 and 14 in the PrE-I region that differ between WT SV5-GFP and the Le-(U5C, A14G) mutant are indicated. le, leader; tr, trailer.
Cells, viruses, and growth analysis.Monolayer cultures of A549 and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum. A549 cells that constitutively express reovirus type 3 Dearing sigma3 protein were generated by transfection with pCXN-S4T3D (25), followed by selection in DMEM containing 0.5 mg/ml G418. Individual colonies were picked, expanded in media containing G418, and screened by Western blotting with polyclonal rabbit serum against sigma3. The sigma3 plasmid and antiserum were kindly provided by T. Kobayashi and T. Dermody (Vanderbilt University School of Medicine).
WT SV5 expressing green fluorescent protein (SV5-GFP) was recovered, as described previously (46), from a cDNA plasmid (16) kindly provided by Robert Lamb (Northwestern University) and Biao He (Penn State University). SV5-GFP stocks were grown in MDBK cells. To construct the SV5 leader mutant Le-(U5C, A14G), PCR was used to introduce a T-to-C change in position 5 and an A-to-G change in position 14 of the leader region of the infectious clone for WT SV5-GFP. The Le-(U5C, A14G) mutant was recovered as described previously (46), with minor modifications.
Virus stock was generated in Vero cells by low-multiplicity of infection (MOI) infection, as described previously (13), in order to prevent generation of defective particles. To assay virus growth, ∼106 A549 cells in six-well dishes were infected at an MOI of 0.05 (multistep growth) or 10 (single-step growth) for 1 h and replaced with DMEM containing 2% fetal bovine serum. Supernatant was collected at various times, and plaque assays were performed with CV-1 cells as described previously (46).
Western blotting and metabolic labeling.For Western blotting, six-well dishes of cells were infected as described in the figure legends. At each time point, cells were washed with phosphate-buffered saline and lysed in 1% sodium dodecyl sulfate (SDS). The cell lysate protein concentration was determined by a bicinchoninic acid assay (Pierce Chemicals), and equivalent amounts of protein were analyzed by Western blotting with rabbit antiserum to cellular STAT-1 (Santa Cruz Biotechnology), monoclonal anti-V5 (Invitrogen) for V protein, or rabbit sera specific for the individual SV5 NP, P, and M proteins (9). As a loading control, lysates were also probed with mouse anti-β-actin antibodies (A5316; Sigma). Blots were visualized by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Pierce Chemicals).
To examine rates of protein synthesis, cells were mock infected or infected at a high MOI. At the times postinfection (p.i.) indicated in the figure legends, cells were starved for 25 min with DMEM lacking cysteine and methionine and then radiolabeled for 20 min with 100 μCi/ml Tran[35S]-label or [35S]cysteine for detecting V protein. For chase experiments, cells were radiolabeled and then incubated in nonradioactive media for 3 h and lysed in 1% SDS, and equal amounts of protein were immunoprecipitated using rabbit polyclonal antisera to SV5 NP, P, M, and F proteins as described previously (9).
Analysis of viral RNA and small interfering RNA (siRNA) experiments.The WF-PIV genomic promoter was cloned using 5′ rapid amplification of cDNA ends, according to the manufacturer's protocols (Invitrogen), on purified nucleocapsid-derived RNA isolated from infected cells. Details and primer sequences are available upon request. The resulting PCR-amplified fragment was cloned into a pGEM5 vector and sequenced.
An RNase protection assay (RPA) was carried out using an Ambion RPAIII kit according to the manufacturer's protocols and as previously described (9). To detect mRNA, RNA was collected from mock-infected or high-MOI-infected samples by use of Trizol (Invitrogen). To assay levels of viral genomes, cells were collected in phosphate-buffered saline containing 50 mM EDTA and cell lysates were treated with micrococcal nuclease to digest RNAs not encapsidated by NP (24, 35). RNA was isolated by Trizol extraction.
Riboprobes were generated by in vitro transcription of a linearized plasmid encoding negative-sense M (for mRNA) or positive-sense leader-NP junction sequences (for genomes) in the presence of [α-32P]CTP, as detailed previously (9). Hybridized RNA samples were treated with RNase A/T1, resolved on 9 M urea-6% polyacrylamide gels, visualized by autoradiography, and quantified by phosphorimaging.
For knockdown of RIG-I, A549 cells in 24-well plates were transfected using Transit siQuest (Mirus, Inc.) with siRNA specific for RIG-I (Dharmacon M-0125-01; 100 nM final concentration) or nontarget control RNA (Dharmacon D-001206-13; 100 nM final concentration) according to the manufacturer's instructions and as previously described (13). Forty-eight hours posttransfection, cells were infected as described above.
ELISAs and cell viability assays.Immunoreactive IL-6 (BD Opt EIA; BD Biosciences) or IFN-β (PBL Biomedical Laboratories) in extracellular medium was quantified by a dual-antibody sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocols. To allow comparison between experiments, cytokine levels were determined for the number of cells at the time of infection and values were normalized to the level for 106 cells. Cell viability was measured by using a CellTiter 96 AQueaous One Solution cell proliferation assay (Promega) according to the manufacturer's instructions.
Microscopy.Phase microscopy and fluorescent microscopy were carried out as described previously (50). Analysis of IRF-3 nuclear translocation was performed as detailed elsewhere (13). Images were captured using a QImaging digital camera and processed using Q-capture software. Exposure times were set manually to be constant between samples.
Substitutions in the genomic PrE-I region lead to elevated viral protein and RNA synthesis.cDNA clones for the WF-PIV 3′ genomic promoter region were generated from antigenomic RNA using 5′ rapid amplification of cDNA ends-reverse transcription-PCR on nucleocapsid-associated RNA. As shown in Fig. 1A, the WT SV5 genomic promoter consists of the 18-base 3′-end PrE-I, the 18-base internal PrE-II, and a 54-nucleotide spacer region (32). Compared to the sequence of WT SV5, the sequence of the WF-PIV genomic promoter contained nine nucleotide differences, with most of the variation clustering in the spacer region. One nucleotide difference was in the internal PrE-II domain at position 79, but results with a minigenome system have shown that substitutions in this position do not affect SV5 RNA synthesis (32). Likewise, while the 54-base region between the 3′-end PrE-I and the internal PrE-II showed six nucleotide differences, we have shown previously that this region plays a very minor role in RNA synthesis (24). The WF-PIV PrE-I region contained two nucleotide differences: a U-to-C change at position 5 and an A-to-G change at position 14. These PrE-I differences had the most potential significance, because PrE-I is among the most highly conserved regions in the genomes of related paramyxoviruses (31).
The above-described sequencing results raised the hypothesis that nucleotide differences in the highly conserved PrE-I region would alter viral RNA synthesis. To test this hypothesis, a recombinant SV5 [Le-(U5C, A14G)] was engineered to harbor only the U5C and A14G substitutions in PrE-I, with the remainder of the genome being derived from WT SV5. As shown in Fig. 1B, the Le-(U5C, A14G) mutant also contained a GFP gene inserted between the HN and L genes, as described previously (16). For these studies, WT SV5-GFP (16) was used as the proper control for analysis of the Le-(U5C, A14G) mutant.
Cells infected with the Le-(U5C, A14G) mutant were found to overexpress viral protein. This is evident in the micrographs in Fig. 2A, where GFP expression was enhanced in cells 24 h p.i. with the Le-(U5C, A14G) mutant compared to that seen with WT SV5-GFP. Western blotting analysis revealed similar results for other viral proteins (data not shown). To determine whether increased levels of viral protein correlated with elevated rates of translation, infected cells were radiolabeled with Tran[35S]-label at various times p.i., and proteins were analyzed by immunoprecipitation. As shown in Fig. 2B, the rate of protein synthesis at 16 h p.i. for the Le-(U5C, A14G) mutant was 1.6- to 2.7-fold higher than that for SV5-GFP, and this was seen for proteins encoded in both the 3′-proximal (e.g., NP) and the 3′-distal (e.g., F) regions of the genome. The rate of viral protein synthesis for both SV5-GFP and the Le-(U5C, A14G) mutant decreased at later times p.i. to ∼30 to 40% of that seen at 16 h p.i. However, translation for the mutant was always higher than that seen for SV5-GFP at all time points and for all genes examined.
The Le-(U5C, A14G) mutant overexpresses viral protein compared to WT SV5. (A) Microscopy. A549 cells were mock infected or infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant. Cells were examined by microscopy at 24 h p.i. (B) Rate of viral protein synthesis. A549 cells were mock infected (lane M) or infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant. At the indicated times p.i., cells were radiolabeled for 20 min with Tran[35S]-label. Cells were lysed, and equal amounts of protein were immunoprecipitated with antibodies to the indicated proteins before analysis by SDS-polyacrylamide gel electrophoresis and autoradiography. Numbers beneath the autoradiograms indicate the expression levels relative to those for WT-infected cells at 16 h p.i. (C and D) Virus growth kinetics. A549 cells were infected at an MOI of 10 (C) or 0.05 (D), and virus in the media at the indicated times p.i. was quantitated by plaque assay. Data are representative of three independent experiments.
The Le-(U5C, A14G) mutant grew to a titer ∼1 log higher than that for SV5-GFP under single-step growth conditions (Fig. 2C), but growth of the mutant virus was very similar to that of SV5-GFP by multistep growth analysis (Fig. 2D). As we have reported previously (13), the enhanced gene expression and WT growth properties for the Le-(U5C, A14G) mutant are not consistent with defective viral particles, and the PFU-to-HA ratios were very similar between WT (10,766 ± 1,335) and mutant (8,723 ± 1,897) viruses.
Levels of viral protein expression for negative-strand RNA viruses are thought to be regulated primarily at the level of transcription (26). To determine whether levels of viral RNA were also altered for the Le-(U5C, A14G) mutant, RPAs were carried out using riboprobes specific for viral genomic RNA or for M mRNA as a representative viral gene. As shown in Fig. 3, M mRNA levels in cells infected with SV5-GFP were initially high at 18 h p.i., but levels decreased to the level of detection by 48 h p.i. This result is consistent with the decrease in translation rates shown in Fig. 2B. In contrast, cells infected with the Le-(U5C, A14G) mutant maintained levels of mRNA that were more than twofold higher than those for WT SV5 at all times p.i. In contrast to mRNA, levels of genomic RNA were very similar for cells infected with WT and mutant viruses. Genomic RNA remained relatively constant out to 72 h p.i., with the exception of slightly lower levels for the Le-(U5C, A14G) mutant at 72 h p.i., which may reflect the cytopathic effect (see below). Taken together, these results indicate that the Le-(U5C, A14G) mutant overexpresses viral protein, which correlates with an increase in the ratio of viral mRNA to genomic RNA compared to the ratio for WT SV5.
Accumulation of Le-(U5C, A14G) mutant mRNA and genomic RNA. A549 cells were mock infected (lane M) or infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant. At the indicated times p.i., RNA was harvested as described in Materials and Methods and analyzed by an RPA using 32P-labeled riboprobes specific for M mRNA or the leader-NP junction for genomic RNA. − and + indicate control samples where riboprobe alone was incubated without or with nuclease, respectively. The − lanes represent 1/50 of the input probe for each sample. The number for each lane indicates the change in signal (n-fold) relative to the level at 18 h p.i., with the level for SV5-GFP set at 1.0.
The Le-(U5C, A14G) mutant induces cytokine responses, despite expressing a functional V protein.The above-described finding of elevated viral mRNA synthesis with the leader mutant raised the hypothesis that the Le-(U5C, A14G) virus would induce antiviral responses. To test this hypothesis, A549 cells were mock infected or infected at a high MOI with SV5-GFP or the Le-(U5C, A14G) mutant, and levels of secreted IL-6 and IFN-β were examined by ELISA. As shown in Fig. 4A, cells infected with the Le-(U5C, A14G) mutant showed a time-dependent increase in IL-6 secretion, which was not seen with SV5-GFP infection. IL-6 induction was eliminated by treatment of the leader mutant virus with UV light (Fig. 4B). Likewise, Le-(U5C, A14G) infection resulted in IFN-β secretion, but unlike with IL-6, the levels of secreted IFN-β were relatively low and did not increase further after 24 h p.i. (Fig. 4C). This IFN induction did not affect low multicycle growth (Fig. 2), consistent with previous work showing that multicycle growth of SV5 expressing a WT V protein is relatively insensitive to low levels of IFN (47).
Induction of proinflammatory cytokines and IFN by the Le-(U5C, A14G) mutant but not by WT SV5. (A, B, and C) Induction of cytokines. A549 cells were mock infected or infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant, and at the indicated times p.i., levels of secreted IL-6 (A and B) or IFN-β (C) were determined by ELISA. Results are the expressed as mean values from triplicate samples, with error bars representing standard deviations, and are normalized to the level for 106 cells. *, P value of <0.005 compared to corresponding samples from control cells. For panel B, the Le-(U5C, A14G) mutant virus was treated with (+) or without (−) UV light prior to infection. (D) IRF-3 nuclear translocation. A549 cells were infected at an MOI of 10 with the indicated viruses. At 24 h p.i., cells were stained for IRF-3 using a monoclonal antibody and for the nucleus using DAPI (4′,6-diamidino-2-phenylindole).
IFN-β synthesis requires the activation and translocation of IRF-3 to the nucleus to initiate transcription of the IFN-β gene (18). To determine the extent of IRF-3 activation by the leader mutant, A549 cells were infected at a high MOI with WT SV5-GFP, the Le-(U5C, A14G) mutant, or SV5-P/V-CPI-, a mutant that we have previously shown to be a potent inducer of IFN-β (9). As shown in Fig. 4D, the diffuse cytoplasmic IRF-3 location in WT SV5-GFP-infected cells resulted in a low labeling intensity that was indistinguishable from that with mock-infected cells, as described previously (9). In the case of the positive-control P/V-CPI- infection, nearly all cells showed intense labeling resulting from translocation of IRF-3 to the nucleus. This is consistent with previous results showing this mutant virus inducing ∼4,000 pg/ml of IFN-β at 24 h p.i. (9). Most of the cells infected with the Le-(U5C, A14G) mutant showed diffuse cytoplasmic IRF-3 staining, although a few cells in the population showed bright nuclear localization. These data are consistent with the results in Fig. 4C showing a low-level induction of IFN-β by the Le-(U5C, A14G) mutant.
The SV5 V protein has been shown to block induction of both IL-6 and IFN-β (17, 27) and can target STAT1 for degradation (8). The backbone of the Le-(U5C, A14G) mutant encodes a WT V protein. As shown in Fig. 5A, the kinetics of V protein accumulation were slightly higher for cells infected with the Le-(U5C, A14G) mutant than for cells infected with SV5-GFP. Consistent with this, immunoprecipitation of lysates from radiolabeled cells showed that the rate of V protein synthesis was elevated for the Le-(U5C, A14G) mutant compared to that for SV5-GFP (Fig. 5B). To confirm that V protein functioned in the context of the Le-(U5C, A14G) mutant infection, A549 cells were infected at an MOI of 10 with the viruses indicated in Fig. 5C, and levels of STAT1 were analyzed by Western blotting at 8 h p.i. This time point was chosen in order to detect any small differences in the STAT1 levels that may not be evident at later times p.i. WT SV5-GFP showed reduced STAT1 levels compared to those for mock-infected cells. STAT1 levels were not decreased following infection with the P/V mutant P/V-CPI-, which is defective in targeting STAT1 degradation (46). Cells infected with the Le-(U5C, A14G) mutant showed a loss of STAT1 equivalent to that seen for WT SV5-GFP. These data indicate that the V protein expression levels are similar to that of WT SV5 and that V protein encoded by the Le-(U5C, A14G) mutant retains function in blocking antiviral signaling.
The Le-(U5C, A14G) mutant (Mut) encodes a functional V protein. (A) Time course of V protein expression. A549 cells were infected at a high MOI, and at the indicated times p.i., cell lysates were analyzed by Western blotting for levels of V protein and cellular actin. (B) Cells infected with the indicated viruses were labeled at 24 h p.i. for 30 min with [35S]cysteine before immunoprecipitation with anti-V protein antibody. (C) STAT1 levels. A549 cells were infected at an MOI of 10 with the indicated viruses. Cell lysates were prepared at 8 h p.i., and levels of STAT1 and actin were determined by Western blotting.
Role of RIG-I and dsRNA in cytokine induction by the Le-(U5C, A14G) mutant.The V protein has been shown to bind to and inhibit the RNA helicase mda-5, but not RIG-I (1, 4). The above-described findings that the Le-(U5C,A14G) mutant induced cytokine secretion in the presence of WT V protein raised the hypothesis that RIG-I contributed to IL-6 and IFN induction. siRNA experiments were carried out to determine whether knockdown of RIG-I levels affected the induction of cytokines by Le-(U5C,A14G) infection. Western blotting showed that levels of RIG-I at 48 h posttransfection were reduced by >50% in A549 cells treated with siRNAs specific for RIG-I (Fig. 6A, lane 3) but not in control samples (Fig. 6A, lane 2). At 48 h after siRNA treatment, cells were mock infected or infected with SV5-GFP or Le-(U5C, A14G). Twenty-four hours later, media were collected and cytokine levels determined by ELISA. As shown in Fig. 6B and C, there was a substantial decrease in levels of IL-6 and IFN-β released by 24 h from infected cells in which RIG-I levels were reduced, indicating a role for RIG-I in activation of cytokines by the Le-(U5C, A14G) mutant. Consistent with other published data regarding A549 cells (3), the level of RIG-I knockdown was only ∼60%. We propose that if knockdown were more complete, the levels of cytokine induction would be further decreased.
Role of RIG-I in cytokine induction by the Le-(U5C, A14G) mutant (Mut). (A) RIG-I knockdown. A549 cells were left untreated (lane 1) or transfected with control siRNA (lane 2) or siRNA targeting RIG-I (lane 3). Forty-eight hours posttransfection, levels of RIG-I and actin were assayed by Western blotting. (B and C) Cytokine secretion. A549 cells were transfected with control siRNA or siRNA specific for RIG-I. At 48 h posttransfection, cells were mock infected or infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant, and levels of secreted IL-6 (B) or IFN-β (C) were measured by ELISA at 24 h p.i. Results are expressed as mean values from triplicate samples, with error bars representing standard deviations, and are normalized to the level for 106 cells. *, P value of <0.005 compared to corresponding samples from control cells.
RIG-I can respond to the presence of dsRNA (23). To determine the role of dsRNA in cytokine induction by the Le-(U5C, A14G) mutant, an A549 cell line was engineered to express the reovirus sigma3 protein, which is known to specifically bind dsRNA (Fig. 7A). A549 control and sigma3-expressing cells were mock infected or infected at a high MOI with the Le-(U5C, A14G) mutant, and levels of cytokines were assayed at various times p.i. As shown in Fig. 7B and C, there was a significant decrease in IL-6 and IFN-β secretion by the Le-(U5C, A14G) mutant in sigma3-expresssing A549 cells compared to the level in control A549 cells. In both the RIG-I knockdown experiment (Fig. 6) and the sigma3-expressing cells (Fig. 7), cytokine induction by WT SV5 was lower than that in the control cells. This is consistent with the published proposal (49) that while SV5 is a poor inducer of cytokines, this is not an absolute, and that infection with WT virus does induce a relatively low level of antiviral response.
Role of dsRNA in cytokine induction by the Le-(U5C, A14G) mutant (Mut). (A) A549 cells expressing reovirus sigma3 protein. Control A549 cells or cells that stably express the reovirus sigma3 protein were analyzed by Western blotting for levels of sigma3. (B and C) Cytokine secretion. Control or sigma3-expressing A549 cells were mock infected or infected at an MOI of 10 with the Le-(U5C, A14G) mutant, and levels of secreted IL-6 were measured by ELISA at the indicated times p.i. For panel C, IFN-β levels were determined at 24 h p.i. Results are expressed as mean values from triplicate samples, with error bars representing standard deviations. * and #, P values of <0.005 and <0.05, respectively, compared to corresponding samples from control cells. (D) Viral M mRNA levels in sigma3-expressing cells. Control or sigma3-expressing A549 cells were mock infected or infected at an MOI of 10 with the indicated viruses. RNA harvested at 48 h p.i. was analyzed by an RPA with a probe specific for M mRNA. − and + indicate control samples where riboprobe alone was incubated without or with nuclease, respectively. The − lane represents 1/50 of the input probe for each sample. M, mock; SV5, SV5-GFP; le, Le-(U5C, A14G) mutant.
Reduced cytokine secretion from sigma3-expressing cells could be due to a reduction in levels of viral mRNA. To test this possibility, levels of M mRNA were determined by RPA at 48 h p.i., a time when there was a large increase in cytokine responses. As shown in Fig. 7D, mRNA levels for WT SV5-GFP were at the limit of detection, as demonstrated in Fig. 3 above), and were not altered by sigma3 expression. Importantly, levels of M mRNA for the Le-(U5C, A14G) mutant were not reduced in the sigma3-expressing cells but instead were ∼3-fold higher than those for the control A549 cells. Together, these results indicate that the Le-(U5C, A14G) mutant induces proinflammatory cytokines through a mechanism that involves production of dsRNA and activation of RIG-I signaling.
The Le-(U5C, A14G) mutant induces cytopathic effects that correlate with increased levels of F protein and cell-cell fusion.Cells infected with the Le-(U5C, A14G) mutant showed cytopathic effects. This is evident in the microscopy pictures in Fig. 8A, where WT SV5-GFP established a largely noncytopathic infection even out to 72 h p.i. In contrast, cultures infected with the Le-(U5C, A14G) mutant showed fewer cells, cell rounding, and syncytia starting as early as 48 h p.i. By use of a cell viability assay (Fig. 8B), high-MOI infection with the Le-(U5C, A14G) mutant resulted in a time-dependent loss of viability in both A549 and HeLa cells that was not seen with SV5-GFP infection. The loss of viability in cultures infected with the Le-(U5C, A14G) mutant was slower and less extensive than that for the highly cytopathic infection with the positive-control P/V-CPI- mutant (46).
Cytopathic effect induced by the Le-(U5C, A14G) mutant (Mut) but not WT SV5-GFP. (A) A549 cells were infected at an MOI of 10 with SV5-GFP or the Le-(U5C, A14G) mutant, and microscopy pictures were taken at 48 and 72 h p.i. (B) Cell viability. A549 or HeLa cells were mock infected or infected with the indicated viruses at an MOI of 10. Cell viability was determined at 48, 72, and 96 h p.i. using a cell viability assay. Data are from quadruplicate samples and are expressed as percentages of the values obtained with mock-infected cells. Error bars represent the standard deviations.
Le-(U5C, A14G)-infected cells had high levels of cleaved caspase-3 (data not shown), indicating that apoptotic pathways were activated. To determine whether production of dsRNA by the leader mutant contributed to cytopathic effects, control or sigma3-expressing A549s were mock infected or infected with SV5-GFP or Le-(U5C, A14G) and the viability of the population of cells was monitored at 48, 72, and 96 h p.i. As shown in Fig. 9A, the Le-(U5C, A14G) mutant induced nearly identical losses of cell viability in both control and sigma3-expressing cells. Similarly, cell killing by the Le-(U5C, A14G) mutant was not affected by RIG-I knockdown (not shown).
The Le-(U5C, A14G) mutant (Mut) induces cytopathic effects that correlate with increased levels of F protein and cell-cell fusion. (A) Cell viability. Control or sigma3-expressing A549 cells were mock infected or infected with the indicated viruses at an MOI of 10. Cell viability was determined at 48, 72, and 96 h p.i. using a cell viability assay as described in the legend for Fig. 8. Error bars represent the standard deviations. (B) Microscopy pictures. A549 or BHK cells were mock infected or infected with the indicated viruses at an MOI of 10. Microscopy pictures were taken at 48 h p.i. Arrows indicate syncytium pockets. (C) Rate of F protein expression and cleavage. A549 cells that were mock infected or infected with the indicated viruses were pulse-labeled for 20 min with Tran[35S]-label. Cells were either lysed immediately (pulse lanes, P) or lysed after incubation for 3 h in nonradioactive media (chase lanes, C), immunoprecipitated with anti-F antiserum, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
During the course of these studies, it was observed that cells infected with the Le-(U5C, A14G) mutant had increased levels of cell-cell fusion. As shown in Fig. 9B, both A549 and BHK cells showed relatively little fusion after infection with SV5-GFP, but very large pockets of syncytia were evident in cultures infected with the Le-(U5C, A14G) mutant. Increased cell-cell fusion correlated with increased levels of the SV5 F protein. This is shown in the pulse-chase radiolabeling experiment with results illustrated in Fig. 9C, where cells infected with the Le-(U5C, A14G) mutant synthesized ∼2- to 3-fold-higher levels of F protein than cells infected with WT SV5-GFP. Likewise, after a 3-h chase period, elevated levels of F were transported through the Golgi body, as assayed by the cleavage of F0 into F1. Together, these data support the contention that cells infected with the Le-(U5C, A14G) mutant have higher levels of cleaved F protein as a result of the global increase in viral gene expression and that this leads to increased cell-cell fusion and cell death.
WT SV5 is a poor inducer of host cell antiviral responses, whereas the naturally occurring SV5 variant WF-PIV induces proinflammatory cytokines, IFN-β, and low levels of cell killing (6, 17, 46). The present study was initiated by our finding that SV5 and WF-PIV differ in the sequences of their 3′-end genomic promoters. Based on the finding that the 18-base PrE-I is the most highly conserved stretch of nucleotides in the rubulavirus genome (31), we have tested the hypothesis that SV5 harboring these PrE-I changes would display an altered RNA synthesis profile. Introducing the WF-PIV substitutions into the SV5 genomic promoter enhanced viral mRNA and protein ∼2-fold but had no apparent effect on genome replication levels. However, our most striking finding was that the leader mutant virus was a strong activator of cytokine secretion and cell killing, despite expressing a functional V protein antagonist. As detailed below, our data support the proposal that the paramyxovirus genomic promoter can be an important determinant of host cell antiviral responses and cell killing.
A priori, it was not clear what effect incorporating the WF-PIV substitutions into the highly conserved region of the WT SV5 promoter would have on RNA synthesis. For many negative-strand viruses, changes to the leader sequence have been shown to decrease viral RNA synthesis. For example, with the HPIV3 minigenome system, it has been reported that changes made in various positions of the first 16 bases of the leader RNA result in decreased reporter gene expression (19). Likewise, single point mutations made for each of the 3′-terminal 22 nucleotides of the rinderpest virus genome all resulted in lower levels of CAT expression in a minigenome system (30). We have taken a different approach, which is based on infectious virus and the use of naturally occurring variability in the genomic promoter.
Unexpectedly, the Le-(U5C, A14G) mutant accumulated elevated levels of viral mRNA, but the accumulation of viral genomes was not significantly changed over that seen with WT SV5. Thus, enhanced mRNA levels appeared to reflect elevated transcription, which was not an indirect effect of having an increased number of genomic RNA templates. This result contrasts with results for our previously described P/V mutant, which directs ∼10-fold-higher levels of both mRNA and genomic RNA (9, 46). Current models for viral determinants of transcription versus replication include the kinetics of NP accumulation and the binding of NP to the nascent leader RNA (26). However, results with our leader mutant are consistent with previous proposals that additional factors, such as cis-acting sequences, can dictate the balance between transcription and replication (12). A detailed mutational analysis of the SV5 PrE-I region using our established minigenome system (32, 40) should reveal the cis-acting determinants for transcription versus replication.
The most striking finding from our work is that two nucleotide substitutions in the genomic promoter convert WT SV5 into a virus that activates host cell responses. Our results with siRNA knockdown and expression of the reovirus sigma3 protein indicate that cytokine responses are activated through a cellular pathway involving RIG-I and dsRNA. RIG-I is activated by RNA containing a 5′ triphosphate (20), but recent evidence has shown that RIG-I also has specificity for short dsRNAs (23). Thus, our results are consistent with a model whereby changes in the SV5 genomic promoter lead to aberrant synthesis of short RNAs that have at least partial double-stranded properties and/or contain an accessible 5′ triphosphate. There are three possible sources of viral RNA with these properties that could activate RIG-I. One source could be the free 55-base leader RNA which is uncapped, has a 5′ triphosphate, and also may contain dsRNA structure. Previous data from transfection experiments have shown that the measles virus leader RNA can activate RIG-I (37). Second, elevated viral transcription could yield mRNAs with incomplete capping, which would yield 5′-triphosphate mRNAs with dsRNA structure. Finally, it is possible that changes to the genomic promoter lead to increased levels of aborted genome replication products that are inefficiently encapsidated by NP. The most straightforward hypothesis for RIG-I activation by the Le-(U5C, A14G) mutant involves elevated transcription from the viral genome to produce higher levels of free leader RNA, but further studies are needed to test this hypothesis.
It has been reported that the cellular protein La binds to the leader RNA of rinderpest virus (39) and of respiratory syncytial virus (RSV) (3). In the case of RSV, it is proposed that La binding to leader RNA masks recognition by RIG-I (3), although other reports have indicated a role for RIG-I in early responses to RSV infection (28). If La protein is also involved in SV5 infections, it is possible that elevated transcription from the Le-(U5C, A14G) mutant results in inefficient binding by La protein either due to an excess of leader RNA or due to alterations to La binding sites on the leader RNA. Work is in progress to determine whether SV5 produces a free leader RNA in infected cells and what type of role La protein has in RIG-I activation by the Le-(U5C, A14G) mutant.
Our data indicate that the Le-(U5C, A14G) mutant produces levels of dsRNA and RIG-I activation that are not normally seen in the case of WT SV5-infected cells. Consistent with this model, cytokines are induced in cells that are coinfected with both WT SV5-GFP and the Le-(U5C, A14G) mutant (not shown). Using immunofluorescence with an anti-dsRNA antibody, we have been unable to detect increased levels of dsRNA in cells infected with the Le-(U5C, A14G) mutant (not shown). This is consistent with the work of Weber et al. (48), which did not detect dsRNA during infections with negative-strand RNA viruses. Thus, the inhibition of host cell responses in cells expressing the dsRNA-binding protein likely reflects a low level of synthesis of dsRNA from the Le-(U5C, A14G) mutant, which can be sequestered by sigma3. An online RNAfold program (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ) predicts that the 55-base Le-(U5C, A14G) leader RNA folds into a different secondary structure than does WT leader RNA (not shown), although the free energy of folding is not significantly different. Thus, the differential structure of free leader RNA or interactions with RNA binding proteins could form the basis for differences in host cell response to WT SV5 and Le-(U5C, A14G) mutant infections.
There are a number of cellular pattern recognition receptors other than RIG-I that recognize dsRNA to induce host cell responses, including mda-5 and protein kinase R (PKR). The Le-(U5C, A14G) mutant expresses a functional WT V protein with kinetics that match that of the parental WT SV5 virus, making it reasonable to assume that mda-5 is not involved in responses to the leader mutant. Le-(U5C, A14G) infection does not significantly activate PKR or induce a shutdown of translation (data not shown), two properties of a previously described SV5 P/V mutant (13). This differential effect of Le-(U5C, A14G) on activation of RIG-I pathways but not PKR could reflect the relative sensitivities of these two cytoplasmic receptors to levels or lengths of dsRNA, with RIG-I being more easily activated than PKR. Similarly, the Le-(U5C, A14G) mutant induces apoptotic markers, but cell killing by the mutant was not affected by expression of sigma3 (Fig. 8A). Together, these results are consistent with a model whereby cytokine responses to the leader mutant are induced by a RIG-I-dependent pathway that is activated by dsRNA but whereby apoptosis is either independent of dsRNA or more sensitive to very low levels of dsRNA that have escaped sequestering by the dsRNA-binding protein sigma3.
The Le-(U5C, A14G) mutant induced a cytopathic effect that was not seen in cells infected with WT SV5, and infected cells showed increased levels of cell-cell fusion. The SV5 F protein is constitutively cleaved into an active form due to the presence of five arginine residues at the cleavage site between F1 and F2 (34). The mechanisms that control paramyxovirus fusion activity are not completely understood, but F expression level has been shown to be an important parameter (11). Consistent with this, the leader mutant showed ∼2- to 3-fold-higher levels of F protein synthesis as part of the global increase in gene expression across the Le-(U5C, A14G) genome. We propose that the SV5 genomic promoter has evolved to function at a level that is lower than maximal in order to prevent overexpression of potentially toxic gene products, such as the F protein. Our proposal for the genomic promoter is consistent with previous data whereby changes in mRNA start frequency for the SeV F gene lead to increased fusion (22).
In summary, our data support a model whereby the viral genomic promoter plays two important roles in allowing SV5 to limit antiviral responses and cell killing through (i) controlling the production of aberrant viral RNA that can trigger RIG-I-mediated antiviral pathways and also (ii) attenuating viral mRNA transcription across the genome in order to avoid overproduction of potentially toxic gene products, such as the F protein. Controlling production of dsRNA from the genomic promoter may be particularly important for viruses, such as SV5, that express a V protein targeting mda-5 but do not encode antagonists, such as the SeV C proteins, that specifically target RIG-I (42). Likewise, attenuating promoter activity could be an important property of viruses, such as SV5, which can establish highly productive long-term infections with only minimal cytopathic effects.
We thank members of the Parks lab and Doug Lyles for helpful comments on the manuscript. We are grateful to Biao He and Robert A. Lamb (Northwestern University) for the original gift of the SV5 infectious clone and Takeshi Kobayashi and Terry Dermody (Vanderbilt University) for the kind gift of sigma3 plasmid and antiserum.
This work was supported by NIH grant AI-42023.
Received 22 May 2009.
Accepted 2 July 2009.
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Journal of Virology Aug 2009, 83 (18) 9057-9067; DOI: 10.1128/JVI.01055-09
You are going to email the following Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing
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Immunity, Innate
parainfluenza virus 5
Promoter Regions, Genetic


In his biography of Handel, Paul Henry Lang quotes Chrysander on the division of operatic content during The Royal Academy's activity prior to its collapse in 1728. Of 428 operatic performances, Handel's works were represented 245 times; Bononcini, 108; Ariosti, 55; and all others, 79. Lawrence Zazzo didn't come to the idea for an album devoted to the Academy's operatic triumvirate from Lang, but according to his release notes, from very favorable contemporary comments about Bononcini and Ariosti. The Handel revival has done little to help their reputations, and that led to a search for original manuscript copies, as opposed to the usual arie antiche arrangements of such pieces as "Per la gloria d'adoravi" (Bononcini's Griselda). Much attention went in the 18 selections of The Royal Trio to presenting Handel's rivals at their most favorable and characteristic, alongside Handel's own work from that period.
The results are entertaining, in particular Bononcini's light touch and gift for easily memorable melody. But lacking the theatrical and musical context of a full performance, such numbers as the pair forming the prison scene from Ariosti's Coriolano can only hint at the full power of the opera. Not that I'm ungrateful for the glimpse, but as "Va tacito" shows, a good amount of the effect comes from understanding the text's strategic placement in the libretto, and its musical placement between numbers to either side.
Lawrence Zazzo's performances in general have been well received by critics here at Fanfare over more than a decade, including myself. Still, he's been very active on stage and records for some time, and is currently in his mid-40s. I had my hopes for this album, but also my concerns, as it's not unusual for even the most astute of singers to show some vocal wear and tear by that time in their careers. Both hopes and concerns, as it turned out, were warranted to a degree in this release.
The positives, first. A few low notes to one side, where the voice suddenly shifts into tenor, Zazzo's alto maintains its tonal evenness throughout its range. His enunciation remains good, and that's important because one of his strong points has long been his ability to make much of an aria's or recitative's expressive content of through heightening consonants and vowels. Zazzo isn't a singer who gives an impression of emotional inertness. The relatively quick tempo set by Bates for that Ariosti prison scene mentioned earlier hobbles its theatricality, but there's never any doubt what Coriolano is feeling during it, thanks to Zazzo's interpretation. This carries across into the slower arias as well, where phrases spaciously, spinning a bel canto line as he varies dynamics.
At the same time, the quantity of slow and medium tempo arias perhaps demonstrates an awareness that the extremely fast fireworks pieces aren't the most successful things on the album. (And even one of the faster arias, "Freme l'onda" from Ariosti's Il naufragio vicino, just leaves the singer commenting briefly at several points, with only some moderate coloratura towards its conclusion.) A piece such as "Vivi, tiranno" (Handel's Rodelinda) has some sketchy and tonally insecure fioritura, while "Tigre piagata" (Bononcini's Muzio Scevola) has some approximate pitching and a loss of breath. He trills well, and can vary both his vibrato and its intensity, but lacks the metal for a hunt-like number such as "Torrente che scende" (Bononcini's Crispo).
Even "Per la gloria d'adoravi," despite tasteful decorations and some subtle dynamics, ends up too loud at phrase endings, too indelicate to score its points. At its best in "Cosi stanco Pellegrino" (Bononcini's Vespasiano), "Chiudetevi niei lumi (Handel's Admeto), and "Tanti affanni" (Handel's Ottone), this album gives us more than a glimpse of a fine dramatic artist who knows how to shape arias carefully and to maximum effect. I could only wish more of it was on that level.
Earlier I criticized David Bates for pushing the pace in Coriolano's prison scene. Much the same can be said of "Per la gloria d'adoravi," where it might have had a negative impact on Zazzo's phrasing. On the other hand, a quicker tempo works to the advantage of "Va tacito," where sharp accenting of the meter and La Nuova Musica's precision supplies less an impression of weight than several other performances, and more of treading through danger. The horns are exceptionally good here; and given the quality of the orchestra, their four overtures and sinfonias are welcome in the album.
A decidedly mixed reaction, then, to A Royal Trio. It should, if nothing else, create some interest in the music of Handel's main operatic rivals at The Royal Academy. But I can't help wishing it had been made with a fresher voiced Zazzo, and perhaps with other artists to provide some tonal variety. Recommended, with reservations.


Make-do Mum: Real Groovy is shutting down in Wellington - vinyl craft.
Real Groovy is shutting down in Wellington - vinyl craft.
Vincent and I had some time off together this morning. With the four year old at creche, and the baby nodding off in the front pack, it felt like we were on a care-free holiday. It is amazing what passes for a holiday when you have children. Our aim was to get to Global Fabrics for their one day sale (so much beautiful fabric) but mainly we just bummed along Cuba Street.
We hadn't gotten very far when we saw Real Groovy records. They are shutting down, like many other record stores, as the economies of selling phyiscal vinyl records, second hand CDs and even new CDs just don't work these days.
There were many sad vinyl records - and many that we knew people would not be buying - we decided to turn these into a craft opportunity. We bought 40 small vinyl records (at 10c each this was not a great expense) for our daughter's creche to turn into an art activity. We then bought 20 large records as the album covers make great envelopes for holding children's art (again for the creche). We found two Madame Butterfly albums with beautiful artwork - these will go on our walls.
I found tons of cool art activities to do with your old records, so thought that I would bring up the very sad closing down sale of Real Groovy, in the hope that you may like to relieve them of their burden of records.


My background is in Marketing, PR and Journalism so I have spent my adult life talking to clients and writing their stories. Now I'd like to help you live the best version of your life.
Cognitive Hypnotherapy has helped me navigate through a series of challenges – bereavement, serious illness, family/teenage issues, divorce – all the usual stuff we all encounter along the way. Talking to a Cognitive Hypnotherapist led me to see things in a new light and put my life back on track. In fact it gave me a new track altogether. It helped me realise that any future we envisage for ourselves is a fiction – so why spend time worrying about what might or might not happen, about things we can't control? We all have the capabilities within us to solve our own problems or issues but sometimes we just need a bit of help to point us towards where to go to find these resources.
I received my diploma from the renowned Quest Institute and am currently undertaking the Master Practitioner programme. I am a member of the QCHPA, the NCH and the CNHC and am bound by their codes of ethics. I am also an NLP Practitioner and now work out of the Mind Studio in Amersham, a dynamic, supportive and inspiring place for change.


text:
Chinese Savers in a Currency Debasement Framework
On more than one occasion, we have explained that compared to Western savers (who have flaws of their own, of course), Chinese savers are let's say less sophisticated for a wide range of reasons. Before continuing with this article, it therefore makes sense to enumerate the most important reasons and take it from there. Please note that this list by no means intends to be definitive, think of it as merely a starting point:
The fact that China's education system is still far from optimal, despite the fact that impressive progress has been made. In many regions of China, there are still issues with basic literacy and as such, expecting the average Chinese saver to properly understand today's ultra-complex realities would be unrealistic. For more information about China's education system and how it compares to various Western counterparts, we would recommend reading the article we have dedicated to this topic by clicking HERE
The fact that various dimensions of China's financial system tend to have an above-average percentage involvement of so-called "retail" investors compared to professionals, with the Chinese stock market being a relevant example in that direction. Once again, we have dedicated an entire article to this topic, one which can be found HERE
The fact that the average Chinese citizen doesn't exactly have as much access to let's call it "less than curated" investment information. Say what you will about Western financial media outlets but compared to the information level of the average Chinese investor, Westerners seem light years ahead. While it is true that gaining access to the "real" Web by for example bypassing the Great Chinese Firewall is hardly impossible, it is fairly safe to assume that Chinese savers are at a disadvantage compared to their Western counterparts
The fact that from a cultural perspective, there is hardly anything in the way of tradition that has been reinforced historically speaking. To put it differently, older generations have been far too "busy" with subsistence to care all that much about being wise savers, with them hardly having enough wealth at their disposal for dedicating a lot of time to such endeavors to make sense
The list could continue indefinitely but the bottom line is this: Chinese savers can, at this point in time at least, be considered at a disadvantage compared to their Western counterparts. However, make no mistake, this most definitely does not mean that Western savers have it easy. On the contrary, as discussed in other articles as well, savers in general have been systematically punished all over the world due to "paradox of thrift" implications: simply put, the worldwide economic system needs perpetual debt-fueled consumerism to sustain itself and savers stand in the way. This, however, is a topic that goes beyond the scope of our article.
Suffice it to say that the current status quo revolves around governments and central banks from all over the world (with China not representing an exception) engaging in currency debasement one can consider unprecedented in terms of sheer contagion (to use a term more and more common in 2020). To put it differently, one cannot exactly seek refuge in Country B's currency if Country A is in full-on debasement mode for the simple reason that the Country B's of the world are engaging in precisely the same behavior. As such, even for sophisticated savers, protecting their purchasing power at the very least and especially enhancing their wealth becomes a problematic goal, with many of them feeling as if they have absolutely nowhere to hide.
Fortunately, this is not true. In fact, we have dedicated a fair number of article to explaining, from A to Z, what savers from all over the world can and should do to protect themselves, with many more articles on the way. Unfortunately, doing so is anything but easy and from the perspective of an unsophisticated Chinese saver who is looking for a "quick fix" solution, the entire equation seems unbelievably confusing.
The end result is therefore likely to involve an extremely large transfer of wealth from those who are not properly prepared/hedged to those who are. Historically speaking, the average citizen is usually on the losing end of such transfers of wealth and the less sophisticated they are, the more affected they are likely to be.
The implications of this reality should be more than obvious: the fact that the proverbial Chinese dream may end up being under siege. Long gone are the pre-2010 days of what seemed to be sustainable double-digit GDP growth, with straightforward international trade dynamics and much-needed domestic infrastructure spending. Fast-forward to the 2020 framework and we have even the Communist Party of China admitting that double-digit growth represents an unrealistic goal, major trade tensions between China and many trading partners that are so frustrated with the trade deficits their countries keep experiencing that they consider China the spoiled child of globalization and infrastructure-driven growth which can only take you so far… an amazingly complicated equation.
To state that the average Chinese saver feels overwhelmed would be a severe understatement and if history is to be any indicator, more or less obvious forms of civil unrest become pretty much unavoidable. As it was, many citizens were frustrated with the various inequality problems that sprung up in the aftermath of China's economic growth story and while more recent administrations such as the Hu Jintao and nowadays Xi Jinping ones have done more than past administrations to tackle this issue, it becomes abundantly clear that innovative solutions are required in order to preserve the "socialism with Chinese characteristics" status quo. Because, make no mistake, a hefty price will need to be paid if the average Chinese saver becomes discontent with the status quo in a manner that leads to frustrations piling up, one the current Chinese administration can most likely not afford, especially given the "tricky" international context that dominates public discourse pretty much all over the world.
Types of Wealth from the Perspective of Chinese Asset Investors
Land Investments in China… and Beyond
Andrei Polgar, the managing partner of ChinaFund.com, is happy to be of assistance and put his expertise as an economist and Wall Street Journal as well as USA Today best-selling author at the disposal of Chinese investment opportunity enthusiasts! Together with experienced entrepreneur Graham Haynes (with brick&mortar success stories in China as well as the United Kingdom) and James MacRae, an accountant with stellar credentials, he forms a remarkably balanced team which can be at your disposal.
How Can ChinaFund.com Help?
While our main goals currently revolve around publishing perspective-altering articles on Chinese investment opportunities, our China-related ambitions don't stop here, simply get in touch to find out how we can help and whether or not our goals align. From macroeconomic expertise to the brick&mortar experience we've gained after 17 years of operation in China, with long-standing government as well as commercial contracts, we're in a unique position to meaningfully understand the Chinese market and help you do the same.
Contact Andrei Polgar
Founding Partner, ChinaFund.com
Shanghai Fanyi Industry & Trade Co., Ltd.
907, West District of Modem Transportation Building, No.218 Hengfeng Road, Zhabei District
Shanghai, China. PC: 200070
contact@chinafund.com
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redpajama_set_name: RedPajamaCommonCrawl


We know her, love her and envy her tan! With hype soaring over the last 6 months, fashion influencer Elle Ferguson has just launched the 'Elle Effect' a tanning kit consisting of a tanning mousse, mitt and stickers so you can customise your bottle. In this blog post I will share my thoughts and before and after pics to show you how I got the Elle Effect!
Summer is less than a month away and my pasty skin is in desperate need of some colour, stat! Thankfully with so many fabulous formulas on the market we don't need to jeopardise our health or skin for the sake of a tan!
In this post I am sharing some of my favourite self, gradual and temporary tanners that I love to use to faux my summer glow!
My trip to Bali is right around the corner and I definitely didn't want to be the palest chick at the beach so I had to call in for reinforcements!
Luckily my friends at Uni Organics had my back and sent me their Forever Tan - Gradual Tan Moisturiser to try out and it has literally allowed me to faux that summer glow!


text:
import os

import numpy as np
import pytest

import pandas as pd
from pandas import DataFrame
import pandas._testing as tm

from pandas.io.excel import ExcelWriter, _OpenpyxlWriter

openpyxl = pytest.importorskip("openpyxl")

pytestmark = pytest.mark.parametrize("ext", [".xlsx"])

def test_to_excel_styleconverter(ext):
    from openpyxl import styles

    hstyle = {
        "font": {"color": "00FF0000", "bold": True},
        "borders": {"top": "thin", "right": "thin", "bottom": "thin", "left": "thin"},
        "alignment": {"horizontal": "center", "vertical": "top"},
        "fill": {"patternType": "solid", "fgColor": {"rgb": "006666FF", "tint": 0.3}},
        "number_format": {"format_code": "0.00"},
        "protection": {"locked": True, "hidden": False},
    }

    font_color = styles.Color("00FF0000")
    font = styles.Font(bold=True, color=font_color)
    side = styles.Side(style=styles.borders.BORDER_THIN)
    border = styles.Border(top=side, right=side, bottom=side, left=side)
    alignment = styles.Alignment(horizontal="center", vertical="top")
    fill_color = styles.Color(rgb="006666FF", tint=0.3)
    fill = styles.PatternFill(patternType="solid", fgColor=fill_color)

    number_format = "0.00"

    protection = styles.Protection(locked=True, hidden=False)

    kw = _OpenpyxlWriter._convert_to_style_kwargs(hstyle)
    assert kw["font"] == font
    assert kw["border"] == border
    assert kw["alignment"] == alignment
    assert kw["fill"] == fill
    assert kw["number_format"] == number_format
    assert kw["protection"] == protection

def test_write_cells_merge_styled(ext):
    from pandas.io.formats.excel import ExcelCell

    sheet_name = "merge_styled"

    sty_b1 = {"font": {"color": "00FF0000"}}
    sty_a2 = {"font": {"color": "0000FF00"}}

    initial_cells = [
        ExcelCell(col=1, row=0, val=42, style=sty_b1),
        ExcelCell(col=0, row=1, val=99, style=sty_a2),
    ]

    sty_merged = {"font": {"color": "000000FF", "bold": True}}
    sty_kwargs = _OpenpyxlWriter._convert_to_style_kwargs(sty_merged)
    openpyxl_sty_merged = sty_kwargs["font"]
    merge_cells = [
        ExcelCell(
            col=0, row=0, val="pandas", mergestart=1, mergeend=1, style=sty_merged
        )
    ]

    with tm.ensure_clean(ext) as path:
        writer = _OpenpyxlWriter(path)
        writer.write_cells(initial_cells, sheet_name=sheet_name)
        writer.write_cells(merge_cells, sheet_name=sheet_name)

        wks = writer.sheets[sheet_name]
        xcell_b1 = wks["B1"]
        xcell_a2 = wks["A2"]
        assert xcell_b1.font == openpyxl_sty_merged
        assert xcell_a2.font == openpyxl_sty_merged

@pytest.mark.parametrize(
    "mode,expected", [("w", ["baz"]), ("a", ["foo", "bar", "baz"])]
)
def test_write_append_mode(ext, mode, expected):
    df = DataFrame([1], columns=["baz"])

    with tm.ensure_clean(ext) as f:
        wb = openpyxl.Workbook()
        wb.worksheets[0].title = "foo"
        wb.worksheets[0]["A1"].value = "foo"
        wb.create_sheet("bar")
        wb.worksheets[1]["A1"].value = "bar"
        wb.save(f)

        writer = ExcelWriter(f, engine="openpyxl", mode=mode)
        df.to_excel(writer, sheet_name="baz", index=False)
        writer.save()

        wb2 = openpyxl.load_workbook(f)
        result = [sheet.title for sheet in wb2.worksheets]
        assert result == expected

        for index, cell_value in enumerate(expected):
            assert wb2.worksheets[index]["A1"].value == cell_value

def test_to_excel_with_openpyxl_engine(ext, tmpdir):
    # GH 29854
    df1 = DataFrame({"A": np.linspace(1, 10, 10)})
    df2 = DataFrame({"B": np.linspace(1, 20, 10)})
    df = pd.concat([df1, df2], axis=1)
    styled = df.style.applymap(
        lambda val: "color: %s" % ("red" if val < 0 else "black")
    ).highlight_max()

    filename = tmpdir / "styled.xlsx"
    styled.to_excel(filename, engine="openpyxl")

    assert filename.exists()
    os.remove(filename)

redpajama_set_name: RedPajamaGithub


text:
Q: Can't upload file in PHP using $ajax() I was trying to upload file on server using php with jQuery's ajax() function. Below is the code which I was trying. Things worked fine when I wrote PHP code on same page without jQuery, so there is no doubt that file upload is working.
HTML

<script src="https://ajax.googleapis.com/ajax/libs/jquery/3.4.1/jquery.min.js"></script>
<script>
    $(function() {
        $("form").submit(function(e){
            e.preventDefault();

            var fd = new FormData();
            fd.append("files", $("#fileinput").prop("files"));
            $.ajax({
                url: "imgupload_.php",
                type:"POST",
                processData: false,
                contentType: false,
                data: fd,
                success: function(result){
                    alert(result);
                }
            });
        });
    });
</script>
<form method="POST" action="#" enctype="multipart/form-data">
    <input type='file' name='files' id="fileinput"/>
    <input type='submit' value='Submit' name='submit'/>
</form>

imgupload_.php
if(isset($_POST["submit"])) {
    $target_dir = "images/";
    $target_file = $target_dir . basename($_FILES["files"]["name"]);

    if (move_uploaded_file($_FILES["files"]["tmp_name"], $target_file)) {
        echo "The file ". basename( $_FILES["files"]["name"]). " has been uploaded.";
    } else {
        echo "Sorry, there was an error uploading your file.";
    }
}

If you want any other info please comment below.

A: You're checking if(isset($_POST["submit"])) but this is not set.
Instead of just this:
var fd = new FormData();

Use this to pull in any non-file input values (such as your submit button.)
var fd = new FormData(this);

redpajama_set_name: RedPajamaStackExchange


receptacles are not all the same. For safety's sake, the power lead goes on the terminal that is on the side with the smallest slot.
Plumber Punky commented on museum_guy's reply to LSA Registered Items Shuffled and Lost?
It might be as simple as a "click here to download your tool list" link. It doesnt need to be a dynamic pretty web page.
Plumber Punky replied to whats that in the back?
Good luck with that. It's (mostly) past line-up. You know, stuff they came out with that's obsolete before it hits the shelves.


text:
St Clement, Bankston
St. Joseph's, Farley
St Patrick, Epworth
St. John the Evangelist
Liturgical Minister
Altar Server Booklet
Wedding Handbook
Liturgical Ministers Schedules
Pastorate Gallery
St. John, Placid Gallery
St. John the Baptist Galley
St. Joseph Gallery
St. Patrick Gallery
St. Clement Gallery
Cemetery Guidelines
Pastorate Building/Grounds Rentals
Archdiocese of Dubuque
Senior Friends List
Seton School
Jim Hammerand
by MicroKii Admin | Jan 15, 2014
Jim Hammerand works for St. Elizabeth Pastorate as a Faith Formation Leader working with youth from the Pastorate. Jim and his wife Lynn have 5 boys. Lynn works for Finley Hospital in the operating room. They make their home in rural Epworth.
Jim grew up in the Sherrill, Iowa area on a dairy farm. Upon graduating from Hempstead High School, Jim attained a BS in Dairy Science from Iowa State University. Furthermore, he was brought up Methodist and went through the RCIA program. Additionally, he graduated from Clarke University with a degree in Religious Studies.
Jim has been in his position since the fall of 2012. During that time he has worked to merge two part time positions into his full time role. Jim worked to start our first ever Summer Program in the summer of 2013.
When asked what message he would like to give members of the pastorate, Jim replies: Thanks for the opportunity to work with your kids. The faith formation that you do at home is so important and we will continue to strive to support your efforts.
Welcome to St. Elizabeth Pastorate
Fr. Michael Schueller
Remembering always that our main mission is to proclaim the Gospel to all, the St. Elizabeth Pastorate parishes seek to support the vitality of all its member parishes. The St. Elizabeth Pastorate's mission is to nurture the Catholic faith among all parishioners, while respecting the individuality of all member parishes.
Pastorate Minutes
Our Faith STO
Online Document Center
Online eDocs
© 2021 St.Elizabeth Pastorate. Designed By Mobile City Media Inc.


We help parents save time on finding the information they need. Our mission is to help families spend more time together, and to make the world a kids-friendly place.
Time Out & Activities, Education & Daycare, Restaurants & Cafes, Sports & Active Leisure, Shopping, Healthcare, and Other Services. The list can be filtered by country and city.
we publish photos, comments and feedback written by parents, as well as lengthy and thorough reviews, put together by our expert editors.


I think when we were kids, sensory play was just called play. But it makes sense to look for activities for our kids that really engage their senses to help them learn. I know certainly that many of my memories are based around sensory experiences. There is a particular sushi shop that I walk post and it instantly transports me back through my memories of travelling in Japan.
While walking the aisles of Spotlight one day, I had the idea of creating a pom pom sensory play tub for my toddler. Pom poms come in so many colours, sizes and textures as well as being light and compact making them the ideal solution for small spaces or houses that are already overrun with toys.
I purchased about 10 packets of different pom poms in an assortment of colours, sizes and textures. Each packet was about $2-3 making it quite an inexpensive activity. All you need is a bowl or tub to keep your pom poms in, ideally one with a lid for easy storage without pom poms spilling everywhere, and away you go.
We have so much fun with our pom pom sensory tub. I probably bring it out once a week and can get a good half hour of happy play from Little Miss. It is a slightly messy activity but easy to clean up so no harm done. Part of the joy for me is just running my fingers through the pom poms – it is quite therapeutic for a tired mama!
Do you have any good sensory activities for toddlers? Have you tried a pom pom sensory play table?


This is the last photo of my baby belly during my pregnancy with Squiggle and soon we will meet my little boy. I can't wait!
That 39 1/2 weeks pregnant belly is so big and so round and SOOOOOOO BEAUTIFUL!!!!!!!!!!!!!!!!!
Thank you very much There is nothing more special than creating new life and pregnancy is certainly a one of a kind journey that I have loved to document, particularly for my children when they are older. Pregnant with my third at the moment, and I certainly have a very big, round belly again. Very special time of my life.


In Florida, "deliver" or "delivery" means the actual, constructive, or attempted transfer from one person to another of a controlled substance. These transactions are often done under surveillance and might be video or audio recorded. In many cases, Law Enforcement use "confidential informants" in the transaction and the State's case is built upon the information provided by these individuals.
The unlawful sale of any drug is a felony. Most charges involving the sale of controlled substance are second degree felonies, which means that they are punishable by up to 15 years in prison (this is the statutory maximum). The sale of marijuana and sale of certain pills are third degree felonies and punishable by up to five years in prison (the statutory maximum). As a result of population growth from Stuart to Vero Beach, and the construction of more schools, day care facilities, churches and convenience stores, more and more people wind up with enhanced charges. Florida Statute 893.13 specifically provides for enhancement penalties if the crime occurs within 1,000 feet of a convenience business, place of worship, school, or day care facility. A link to Florida Statute 893.13 is here for your convenience.
The Law Offices of David Golden, P.A. has had great success in defending the rights of individuals charged with the sale or delivery of a controlled substance by either attacking the credibility of the confidential informant or by finding other technical defects in the State's case. Contact the office now at (772) 220-4400 to schedule your FREE CONSULTATION.


text:
Enterprise Informatics Signs Up Two Nuclear Operators
Enterprise Informatics, a San Diego company that sells Microsoft-based management software for industrial clients, signed up a pair of nuclear operators this week for its flagship product, Enterprise Bridge for Nuclear.
Wolf Creek Nuclear Operating Corp. in Kansas and Nuclear Fuel Services in Tennessee both selected Enterprise Bridge for Nuclear to increase efficiency in design engineering and comply with Nuclear Regulatory Commission guidelines, the company said Dec. 3.
Terms of the deals were not disclosed.
Enterprise Bridge is Enterprise Informatics' flagship product. It was developed based on Nuclear Regulatory Commission guidelines to help plants remain compliant with regulatory mandates.
Wolf Creek Nuclear Operating Corp. owns and manages the Wolf Creek Generating Station just northeast of Burlington, Kansas, which generates about 1,200 megawatts , enough energy for approximately 800,000 homes.
Nuclear Fuel Services enriches nuclear fuel, primarily for the Navy. The private company is licensed by the U.S. Nuclear Regulatory Commission and is expanding to provide fuel for the commercial market.
"There are millions of documents underwriting hundreds of thousands of components that need to be properly tracked and stored while being easily accessible for operation use over the life of the plant," said Enterprise Informatics CEO Alan Kiraly. "EB's unique information management approach to asset, document and records management , coupled with enterprise-class server architecture , provides a solution that meets this need in a familiar and affordable Microsoft environment."
Enterprise Informatics serves industries around the globe, including government, utilities, transportation, financial services, engineering and construction.
, Ned Randolph
Artes Files for Chapter 7
One in 10 Homeowners Nationwide Behind on Mortgages


Sockeye continue to pour into the Situk on every incoming tide. Commercial fishing on Sunday through Tuesday creates a slight slow down in numbers, but there is still plenty of opportunities to catch a limit of nice fish.
King populations are still down and any deliberate fishing or retention of any size king on the Situk is illegal at this point.
Pink Salmon are starting to show in very low numbers at this point and the Sockeye fishing should remain stellar throughout the month of July.There have also been reports of a couple early Silvers showing up in the commercial nets.
If you are one of the many individuals that have never heard of Yakutat Alaska you are not alone. Yakutat's isolated population center is located at the mouth of Yakutat Bay with less than 1000 inhabitants. The total area spanning about 9,400 square miles, of which 7,600 square miles is land and 1,800 square miles is water. Fishing is the main industry in the well-protected area and it is an area only accessible by plane or boat. There are no roads to access this secluded wilderness meaning it is a perfect destination to unwind and reconnect with Mother Nature.
The area of Yakutat is bordered on two sides by Canadian Provinces British Columbia and Yukon and two sides by Alaska. There are main sites to take in within this borough as it contains portions of the protected areas of Chugach National Forest, Glacier Bay National Park, Glacier Bay Wilderness, Tongass National Forest, Wrangell-St. Elias National Park and Preserve, Wrangell-Saint Elias Wilderness and the Russell Fjord Wilderness.
One of the many unique features in the Borough is Hubbard Glacier, which is North America's largest tidewater glacier and one of the only glaciers currently increasing in size yearly rather than decreasing in size. Another unique natural feature in the area is that the mountain range located in Yakutat has 2 or 3 of the top 10 highest peaks in the world 10,000+ feet, which adds to the already spectacular backdrop. In addition there is a near by lake called Pike Lake which was formed during the Ice age and has the oldest species of pike on the planet, dating back its origins to the ice age. These pikes look like something from a prehistoric museum and only exist in this one location. Yakutat also has the largest run if wild steelhead in the world, making it famous for steelhead fishing enthusiasts.
The fishing in the area is also unique, having access to world-class fresh and saltwater locations at your finger tips. If you are fishing for Steelhead, Salmon or other freshwater varieties or if you are chasing halibut or rock fish from the depths of the saltwater Yakutat has trophies of all species and sizes. Having access to saltwater and fresh water in one location offers guests and anglers a very unique fishing opportunity which attracts anglers from around the world. Due to the seclusion of the location you can guarantee that you will not be sharing the waters with many others, which is unique considering iYakutat offers some of the best fishing in all of Alaska. Choosing a trip to Yakutat will be sure to nurture solitude and your connection with the great outdoors.
The surrounding area is not only for fishing, there are other attractions to take in. In summer, surfers travel to the great north for bragging rights of surfing the coldest and most elusive waves on earth. Photography enthusiasts travel from all over the world to take in the sites that have not been touched by man. There are few places on earth that have been left for the modern day explorer. This is one of those locations. If you are a bird enthusiasts, this is the perfect place to catch a glimpse of bald eagles of all maturity levels up close. With the bald eagle being one of the nations most respected species to see such an abundance is quite magical.
The Yakutat Lodge is proud to have the comforts of civilization in one of the few remaining untouched and pristine nature reserves in all of the U.S. We are happy to answer any questions you might have in regards to planning your next trip to Yakutat. For more information head to our website www.yakutatlodge.com, email us at info@yakutatlodge.com or call us direct at 1-800-YAKUTAT. We look forward to hearing from you and introducing you to the area, no matter what your tastes are.


This is by far one of my favorite dishes for brunch... and I say brunch because you can enjoy it at either breakfast or lunch. As a new mom, this is also the dish that makes me feel most put-together– it's luxurious, balanced, and powerful start to your day! Best of all, it takes about 3min to prepare. I've remade this recipe at least 3x since having Winnie & will usually freeze 6-8 bowls at a time.
Prepare quinoa and sweet potato in advance. For quinoa cook plain, 1 to 1 ration of quinoa to water. For sweet potatoes wash and cut into 1inch wedges– bake plain.
Dish up kale, quinoa, sweet potatoes (diced), and black beans. To store for later, use quart sized freezer Ziploc & leave some air in the bag– do not pack down. When ready to enjoy, microwave for approx 2min from frozen. In small nonstick pan, fry two whole eggs– I highly recommend pasture raised! Serve over easy for that runny yolk, top with salt, pepper, and hot sauce (optional). Enjoy!