Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir.

Human dengue viruses emerged from primate reservoirs, yet paradoxically dengue does not reach high titers in primate models. This presents a unique opportunity to examine the genetics of spillover versus reservoir hosts. The dengue virus 2 (DENV2) - encoded protease cleaves human STING, reducing type I interferon production and boosting viral titers in humans. We find that both human and sylvatic (reservoir) dengue viruses universally cleave human STING, but not the STING of primates implicated as reservoir species. The special ability of dengue to cleave STING is thus specific to humans and a few closely related ape species. Conversion of residues 78/79 to the human-encoded 'RG' renders all primate (and mouse) STINGs sensitive to viral cleavage. Dengue viruses may have evolved to increase viral titers in the dense and vast human population, while maintaining decreased titers and pathogenicity in the more rare animals that serve as their sustaining reservoir in nature

dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don't have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.</p

Non-human Primate Schlafen11 Inhibits Production of Both Host and Viral Proteins

<div><p>Schlafen11 (encoded by the <i>SLFN11</i> gene) has been shown to inhibit the accumulation of HIV-1 proteins. We show that the <i>SLFN11</i> gene is under positive selection in simian primates and is species-specific in its activity against HIV-1. The activity of human Schlafen11 is relatively weak compared to that of some other primate versions of this protein, with the versions encoded by chimpanzee, orangutan, gibbon, and marmoset being particularly potent inhibitors of HIV-1 protein production. Interestingly, we find that Schlafen11 is functional in the absence of infection and reduces protein production from certain non-viral (GFP) and even host (Vinculin and GAPDH) transcripts. This suggests that Schlafen11 may just generally block protein production from non-codon optimized transcripts. Because Schlafen11 is an interferon-stimulated gene with a broad ability to inhibit protein production from many host and viral transcripts, its role may be to create a general antiviral state in the cell. Interestingly, the strong inhibitors such as marmoset Schlafen11 consistently block protein production better than weak primate Schlafen11 proteins, regardless of the virus or host target being analyzed. Further, we show that the residues to which species-specific differences in Schlafen11 potency map are distinct from residues that have been targeted by positive selection. We speculate that the positive selection of <i>SLFN11</i> could have been driven by a number of different factors, including interaction with one or more viral antagonists that have yet to be identified.</p></div

Schlafen11 reduces protein production from non-viral transcripts.

<p><b>(A)</b> Schlafen11 or Chlor (negative control) expressing plasmids were cotransfected into 293T cells along with plasmids expressing either a V5-tagged GFP or myc-tagged eGFP. Total protein was harvested 48 hours post transfection and probed for the indicated proteins. The blot is representative of 3 independent experiments. <b>(B)</b> Either GFP or eGFP (untagged) expressing plasmids were cotransfected along with plasmids encoding Schlafen11 as in (A). 48 hours post transfection, flow cytometric analysis was performed to measure the mean fluorescence intensity (MFI) of the GFP signal. All values reported are relative the the Chlor control. Error bars are standard error from 3 independent experiments.</p

The <i>SLFN11</i> gene has evolved under positive selection in primates.

<p><b>(A)</b> A phylogeny of the primate <i>SLFN11</i> gene sequences used in this analysis. Sequences obtained from online databases are indicated with asterisks, the others were generated in this study. <b>(B)</b> Table summarizing the likelihood ratio test between the M8 and M8a models in PAML. The 2ΔlnL value (twice the difference in the natural log of the likelihoods) for M8 versus M8a is shown, along with the p-value with which the neutral model M8a is rejected in favor of the model of positive selection. The tree length of the <i>SLFN11</i> gene alignment was 0.65, and 7.6% of codons were assigned a dN/dS = 3.8. PAML analysis was repeated using different codon models (f61, 3x4) and different ω₀ seed values, and in all cases results converged. <b>(C)</b> Residues corresponding to codons with dN/dS > 1 are indicated on a schematic of the Schlafen11 protein.</p

Primate versions of Schlafen11 differentially inhibit retroviral protein production.

<p>Plasmids encoding Schlafen11-V5 from the indicated primate species, or a V5-tagged chloramphenichol acetyltransferase (Chlor) gene as a negative control, were co-transfected into 293T cells along with plasmids encoding <b>(A)</b> a nearly full-length HIV clone (pNL4-3.Luc.R⁺E^-), <b>(B)</b> HIV-1 Gag-Pol-RRE and HIV-1 Rev, <b>(C)</b> MLV Gag-Pol, <b>(D)</b> FIV Gag-Pol. Immunoblotting was used to monitor protein production of chloramphenichol acetyltransferase (Chlor-V5), Schlafen11-V5, GAPDH, and viral proteins. Panels A-D are representative blots from 3 or more experimental replicates. <b>(E)</b> Bands from panels A-D were quantified to show the relative effect of each Schlafen11 homolog. Each experiment was normalized to human Schlafen11. As increasing Schlafen11 activity as seen, the color of the box changes linearly along the blue to red color spectrum (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006066#sec011" target="_blank">materials and methods</a> for quantification procedure). <b>(F)</b> Viruses were packaged in 293T cells in the presence or absence of Schlafen11. Increasing amounts of the plasmids necessary for virus packaging were co-transfected along with a constant amount of plasmid expressing human, gibbon, or marmoset Schlafen11 (or Chlor as a negative control). The resulting virions were then used to infect 2.5x10⁵ 293T cells and the percentage of infected cells was scored by flow cytometry (GFP+). These data are representative of two independent experiments. Herein, “Chlor” is used as an abbreviation instead of the standard “CAT,” since the latter is also the name of another mammal and could therefore cause confusion.</p

Schlafen11 inhibits production of human proteins.

<p><b>(A)</b> Codon adaptation index (CAI) is plotted for all human genes, and for select other genes (viral and GFP/eGFP) used in this study. Human genes analyzed in this study are indicated in red, viral genes in purple. <b>(B,C,D)</b> Plasmids encoding the indicated Schlafen11-V5 proteins, or Chlor, were cotransfected into 293T cells (B) with or without a Vinculin-V5 expressing plasmid, (C) with a GAPDH-V5 expressing plasmid, or (D) with an Actin-V5 expressing plasmid. Cell lysates were subject to immunoblotting as indicated. Blots are representative from two independent experiments. <b>(E)</b> Quantification of the V5-tagged proteins detected in panels B-D. All bands are normalized to the quantification of endogenous GAPDH.</p

Schlafen11 is expressed in various human tissues and is active at physiologically-relevant levels.

<p><b>(A)</b> 293T cells were cotransfected with viral packaging plasmids required to create VSV G-pseudotyped MLV along with increasing amounts of pcDNA6.2 encoding the indicated Schlafen11 or Chlor. Pseudotyped MLV produced was measured on 293T cells and the relative amount of virus production (standardized to Chlor) was determined. <b>(B)</b> Identical to (A), except performed in HUT78 cells (a T-cell line). Only the results from 1000ng is shown as other experiments did not yield replicable results. <b>(C)</b> Identical to (A), except performed in Chinese Hamster Ovary (CHO) cells. <b>(D)</b> Quantitative PCR was used to measure <i>SLFN11</i> expression levels in cDNA from transfection conditions in panels A-C, and from a set of human tissues. Error bars represent the standard error of three independent replicate experiments. <b>(E)</b> <i>SLFN11</i> was amplified by non-quantitative PCR from a cDNA panel representing the human tissues indicated.</p

dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don&rsquo;t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants