A Serpin shapes the extracellular environment to prevent influenza A virus maturation

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response

Regulation of Toll-like Receptor Signaling by the SF3a mRNA Splicing Complex

<div><p>The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.</p></div

SF3a1 inhibition leads to intron retention in several TLR signaling pathway genes.

<p>(A,B) These panels depict sequence reads (average of 3 replicates, each; generated by DEXSeq) across IRAK1 or IKKβ (5’ end of gene on right, control siRNA in red, SF3a1 siRNA in blue). The retained introns (intron 1 in IRAK1 or intron 15 in IKKβ) are shaded in purple. (C-I) These panels display the results of qPCR assays on RAW264.7 cells used to monitor production of the indicated mRNA isoforms (expression normalized so that 1 is the expression in the presence of control siRNA). CT indicates control siRNA in this an all other figures. LPS exposures were performed for six hours in the presence of 20 ng/ml LPS. Asterisks indicate results that were statistically different than control in this and all other figures.</p

Multiple genes in the TLR signaling pathway mediate the effects of SF3a1 on innate immunity.

<p>RAW264.7 macrophages were treated with the indicated siRNAs or control non-targeting siRNA and subsequently exposed to 20 ng/ml LPS for 6 hours. IL-6 production was monitored by ELISA. In panels B and C, cells were treated with multiple siRNAs simultaneously, as indicated; in panel A, only a single siRNA was used in each case.</p

siRNAs targeting IKKβ intron 15 lead to increased production of LPS-induced IL-6.

<p>RAW264.7 macrophages were treated with either of 2 siRNAs targeting IKKβ intron 15 or control non-targeting siRNA, were subsequently exposed to 20 ng/ml for 6 hours, and then expression of both IKKβ isoforms was monitored by qPCR (A,B) and IL-6 production was monitored by ELISA (C).</p

SF3a1 inhibition affects pre-mRNA splicing of many genes in the TLR signaling pathway.

<p>The schematic depicts the Toll-like receptor signaling pathway (KEGG map04620) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref143" target="_blank">143</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref144" target="_blank">144</a>] with genes whose splicing is altered (DEXSeq analysis) when SF3a1 is inhibited color coded as follows: red (altered in the absence or presence of LPS), green (only in the absence of LPS), blue (only in the presence of LPS). Additionally, altered splicing of two genes in purple was identified in our other analyses. Generated using DAVID [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref053" target="_blank">53</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004932#pgen.1004932.ref054" target="_blank">54</a>].</p

Pathways affected by LPS Treatment.

<p>*Innate immune signaling pathways underlined.</p><p>Pathways affected by LPS Treatment.</p

Plan to investigate the effect of SF3a1 inhibition.

<p>The schematic depicts our experimental approach for investigating the effect of SF3a1 inhibition on mRNA splicing and innate immunity. Mouse RAW264.7 cells were treated with either SF3a1 siRNA or control siRNA and subsequently exposed (or not) to LPS. These mRNA samples were then subjected to poly-A mRNA sequencing (A). The sequence data were then analyzed using MISO (B) to determine the global effects of SF3a1 on alternative pre-mRNA splicing and to investigate intron sequences that mediated these alternative splicing effects (C). The sequence data also were analyzed using DESeq, DEXSeq, and Cuffdiff to identify genes and isoforms whose expression was regulated by SF3a1 (B). This gene- and isoform-level analysis was in turn used for pathway analysis (D) and to identify specific TLR signaling pathway genes that mediate the effects of SF3a on innate immunity (E).</p

Identification of intron and exon features that correlate with alternative splicing events regulated by SF3a1.

<p>(A-D) Sequence logo plots of the polypyrimidine tracts and 3’ splice site of introns that are retained when SF3a1 is inhibited (SF3a-dependent) or introns that are spliced out correctly when SF3a1 is inhibited (SF3a-independent, or at least less dependent) in the RAW264.7 cell line. (E) The length of exons that are skipped when SF3a1 is inhibited compared to those exons that are not skipped. The boxplots were generated using default conventions in R 3.0.1. The boxes show the interquartile range, the whiskers the most extreme data point that is no more than 1.5 times the interquartile range from the box. Exon lengths are depicted on a log scale. “Skipped” refers to exons with increased rate of skipping when SF3a1 is inhibited, whereas “not skipped” refers to exons that have the same rate of skipping, regardless of SF3a1 levels.</p

The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

<div><p>The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.</p></div