Effect of TRiP knockdown of <i>Mt2</i> expression in <i>Wolbachia</i> infected flies.

<p><i>Mt2</i> expression was knocked down in <i>Wolbachia</i> infected transgenic RNAi fly stocks 38224 (<i>Mt2</i> shRNA 1) and 42906 (<i>Mt2</i> shRNA 2) by driving <i>Mt2</i> shRNA expression via chromosome II Act5C-Gal4 driver (y¹ w*; P{Act5C-GAL4}25FO1/CyO) as described in Materials and Methods. For each set of crosses, siblings lacking the expression of <i>Mt2</i> targeting shRNA were used as the wild-type controls. (A, B) Flies were challenged with SINV as described previously whereby infection was allowed to last for 48 hours before whole fly tissues were collected and assayed for infectious virus using end-point-dilution assay on BHK-21 cells. Values represent the mean of six independent biological replicates. (C) Quantitative analyses of <i>Mt2</i> expression in TRiP mutant flies relative to their respective wild-type sibling controls (set at 1) was performed using qRT-PCR on total RNA extracted from fly tissues. Values for each column represent the mean of 6 and 3 independent biological replicates, respectively. <i>P</i> values were calculated using Mann-Whitney U tests (<i>Mt2</i> shRNA 1: <i>p < 0</i>.<i>0001</i>, <i>Mt2</i> shRNA 2: <i>p = 0</i>.<i>0361</i>). (D) <i>Wolbachia</i> titer was quantified using qPCR. Reported values are relative to respective wild-type sibling controls (set at 1) and are represented as the mean of two independent biological replicates. All error bars represent standard error of mean (SEM).</p

<i>Wolbachia</i> induced SINV resistance is dependent on host methyltransferase gene <i>Mt2</i>.

<p>(A) <i>Mt2</i> expression profile in <i>Wolbachia</i>–infected (<i>left</i>)and <i>Wolbachia</i>–free (<i>right</i>) flies either in the absence or presence of SINV infection. Virus infection was established as described in Materials and Methods. Infection was allowed to last for 48 hours followed by quantification of <i>Mt2</i> expression via qRT-PCR. <i>P</i> values were calculated using Mann-Whitney U test (<i>p < 0</i>.<i>001</i>) (B) Quantification of infectious virus produced during SINV infection in <i>Wolbachia</i>–infected (<i>left</i>)and <i>Wolbachia</i>–free (<i>right</i>) flies that are either wild-type (<i>WT</i>) or lacking functional <i>Mt2</i> gene (<i>Mt2 -/</i>-). Flies were challenged with SINV for 48 hours before whole fly tissues were harvested and assayed for the presence of infectious virus using end-point-dilution assay in BHK-21 cells. Values represent the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests between genotypes (<i>p < 0</i>.<i>05</i>) (C) Effect of <i>Mt2</i> loss on viral RNA synthesis in the presence of <i>Wolbachia</i>. SINV RNA was quantified using qRT-PCR by probing against the viral E1 gene. Values reported above are relative to viral RNA levels observed in <i>Wolbachia</i>–free wild-type flies. Values represent the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests between genotypes (<i>W+</i>: <i>p < 0</i>.<i>01</i>, <i>W</i>-: <i>p</i> = 0.71) (D) <i>Wolbachia</i>–infected (<i>left</i>)and <i>Wolbachia</i>–free (<i>right)</i> wild-type or <i>Mt2 -/</i>- flies were challenged with SINV cap-nLuc virus. After 48 hours post infection, fly tissues were harvested and assayed for luciferase activity to determine viral structural protein levels. Reported values are relative to nLuc activity observed in wild-type <i>Wolbachia</i>–free (<i>W</i>-) flies, set at 1. Values represent the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests between genotypes (<i>W+</i>: <i>p < 0</i>.<i>001</i>, <i>W</i>-: <i>p = 0</i>.<i>224</i>). All error bars represent standard error of mean (SEM).</p

<i>Mt2</i> antiviral activity does not depend on the <i>Wolbachia</i> infection status of the host.

<p><i>Mt2</i> expression was knocked down in D. <i>melanogaster</i> derived <i>Wolbachia</i>–free JW18 cells using dsRNA against the host methyltransferase gene. Approximately 48-hours post-transfection, cells were challenged with SINV at an MOI of 100 as described in Materials and Methods. Infection was allowed to last for 48 hours before harvesting the cells and media. Total cellular RNA was extracted from cells as described in Materials and Methods and assayed for host <i>Mt2</i> (A) and viral <i>E1</i> (B) gene expression. Values represent the mean of five independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests (<i>Mt2</i>: <i>p < 0</i>.<i>0001</i>, <i>SINV E1</i>: <i>p < 0</i>.<i>0001</i>). (C) Media was collected 48 hours post infection and infectious virus particle was quantified as before, using end-point-dilution assay on BHK-21cells. Values represent the mean of five independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests (TCID50: <i>p < 0</i>.<i>0001</i>). (D) Infectivity of virus particles produced was determined as described earlier, whereby total number of virus particles were quantified using qRT-PCR. As before, higher total particle: TCID50 numbers indicate poorer virus particle infectivity and relative reduction in particle: TCID50 ratio represents an overall increase in infectivity. Values represent the mean of five independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U tests (particle: TCID50: <i>p < 0</i>.<i>0001</i>). All reported values are relative to non-targeting, control dsRNA treatments set at 1. All error bars represent standard error of mean (SEM).</p

<i>Wolbachia</i> reduces SINV RNA synthesis.

<p>(A) Systemic virus infection was established in <i>Wolbachia</i>–infected (<i>left</i>) and <i>Wolbachia</i>–free (<i>right</i>) flies as described in Materials and Methods. Total RNA was isolated from fly tissue homogenates 48 hours post infection and assayed for fold change in RNA synthesis using qRT-PCR. Values reported are the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U test (nsP1: <i>p < 0</i>.<i>0001</i>, E1: <i>p < 0</i>.<i>0001</i>) (B) Absolute quantification of viral plus strand (<i>left</i>) and minus strand (<i>right</i>) RNA species observed during SINV infection in <i>Wolbachia</i>–infected and <i>Wolbachia</i>–free JW18 cells. Cells were infected with SINV at an MOI of 100 that lasted 96 hours, followed by extraction of total cellular RNA. Quantification of SINV RNA species was performed using qRT-PCR as described in Materials and Methods. Values were calculated using standard curves. <i>P</i> values were calculated using Unpaired t-test with Welch’s correction (plus strand: <i>p</i> = 0.043, t = 4.521, df = 2.063, minus strand: <i>p</i> = <i>0.046</i>, t = 4.457, df = 2.012). All error bars represent standard error of mean (SEM).</p

<i>Wolbachia</i> elevates host methyltransferase expression to block an RNA virus early during infection

<div><p><i>Wolbachia pipientis</i> is an intracellular endosymbiont known to confer host resistance against RNA viruses in insects. However, the causal mechanism underlying this antiviral defense remains poorly understood. To this end, we have established a robust arthropod model system to study the tripartite interaction involving Sindbis virus and <i>Wolbachia</i> strain <i>w</i>Mel within its native host, <i>Drosophila melanogaster</i>. By leveraging the power of <i>Drosophila</i> genetics and a parallel, highly tractable <i>D</i>. <i>melanogaster</i> derived JW18 cell culture system, we determined that in addition to reducing infectious virus production, <i>Wolbachia</i> negatively influences Sindbis virus particle infectivity. This is further accompanied by reductions in viral transcript and protein levels. Interestingly, unchanged ratio of proteins to viral RNA copies suggest that <i>Wolbachia</i> likely does not influence the translational efficiency of viral transcripts. Additionally, expression analyses of candidate host genes revealed <i>D</i>. <i>melanogaster</i> methyltransferase gene <i>Mt2</i> as an induced host factor in the presence of <i>Wolbachia</i>. Further characterization of viral resistance in <i>Wolbachia</i>–infected flies lacking functional <i>Mt2</i> revealed partial recovery of virus titer relative to wild-type, accompanied by complete restoration of viral RNA and protein levels, suggesting that <i>Mt2</i> acts at the stage of viral genome replication. Finally, knockdown of <i>Mt2</i> in <i>Wolbachia</i> uninfected JW18 cells resulted in increased virus infectivity, thus demonstrating its previously unknown role as an antiviral factor against Sindbis virus. In conclusion, our findings provide evidence supporting the role of <i>Wolbachia</i>–modulated host factors towards RNA virus resistance in arthropods, alongside establishing <i>Mt2’s</i> novel antiviral function against Sindbis virus in <i>D</i>. <i>melanogaster</i>.</p></div

Effect of <i>Wolbachia</i> on SINV infectivity.

<p>(A) Presence of <i>Wolbachia</i> is correlated with reduced virus titer in <i>D</i>. <i>melanogaster</i> flies. Systemic viral infection was established in <i>Wolbachia</i>-infected (<i>left</i>) and <i>Wolbachia</i>-free (<i>right</i>) flies as described in Materials and Methods. Infection was allowed to last for 48-hours before whole fly tissues were harvested and assayed for the presence of infectious virus using end-point-dilution assay in BHK-21 cells. Numbers along the <i>y</i>-axis represent the dilutions that resulted in at least 50 percent cell death (TCID₅₀). Data are mean values for three independent experiments. <i>P</i> values were calculated using Mann-Whitney U test (<i>p</i> = 0.0050) (B) Presence of <i>Wolbachia</i> in <i>D</i>. <i>melanogaster</i> derived JW18 cells lead to reduced virus particle infectivity over time. <i>Wolbachia</i>-infected (<i>left</i>) and <i>Wolbachia</i>-free (<i>right</i>) JW18 cells were infected with SINV at an MOI of 100 and media was collected at 48, 72 and 96 hours post infection. Samples were subsequently assayed for infectious virus using end-point-dilution assay and for particle numbers by qRT-PCR quantification of viral genome copies present in the media. Higher total particle: TCID₅₀ numbers are indicative of lower virus particle infectivity. Values reported are the mean particle-to-TCID₅₀ ratios of two independent replicates. All error bars represent standard error of mean (SEM).</p

Viral protein synthesis is reduced in the presence of <i>Wolbachia</i> in flies.

<p>(A) <i>Wolbachia</i>–infected and <i>Wolbachia</i>–free flies were each infected with either SINV-nsP3-nLuc (<i>left</i>) or SINV cap-nLuc (<i>right</i>) viruses as described in Materials and Methods. Infection was allowed to last for 48 hours and fly tissues were harvested and assayed for luciferase activity to determine viral nonstructural (<i>left</i>) and structural (<i>right</i>) protein levels respectively. Reported values are relative to nLuc activity observed in <i>Wolbachia</i>–free (<i>W</i>-) flies, each set at a value of 1. Values represented the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U test (non-structural proteins: <i>p < 0</i>.<i>0001</i>, structural proteins: <i>p < 0</i>.<i>0001</i>) (B) Translation efficiency (RLU: RNA) of SINV non-structural (<i>left</i>) and structural (<i>right</i>) polyproteins was calculated as the ratio of total protein (shown above as the net luciferase output or RLU) to absolute copies of viral genomic and subgenomic transcripts, respectively. Absolute quantification of viral genomic and subgenomic RNA species was performed using qRT-PCR as described before. Values represented the mean of three independent biological replicates. <i>P</i> values were calculated using Mann-Whitney U test (non-structural proteins: <i>p = 0</i>.<i>20</i>, structural proteins: <i>p > 0</i>.<i>99</i>). All error bars represent standard error of mean (SEM).</p

Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

<div><p>Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.</p></div

Mutation of the SINV C:R interaction sites negatively affects incoming viral genomic RNA function.

<p><b>A</b>) The RNA decay profiles of the incoming viral genomic RNAs of the individual SINV C:R interaction site mutants and parental wild type virus were determined by qRT-PCR analysis as described in the materials and methods. Plotted is the relative abundance of the incoming viral genomic RNAs (y-axis) with regards to time (x-axis). Regression analysis was utilized to determine the RNA decay profile (as shown with the solid line) and the dashed lines represent the 95% confidence intervals of the aforementioned regression. <b>B</b>) The half-lives of the individual genomic RNAs as determined using the calculations reported in Dolken et al., as determined by the first point at which the relative abundance has reached 0.5. C) The levels of Nanoluciferase activity for wild type parental virus and the nt10100 and nt10400 C:R interaction site mutants were determined as reported in the materials and methods at the indicated times post infection. All quantitative data in this figure represents the mean of at least three independent biological replicates. Comparative analysis was performed using variable bootstrapping, as described in the materials and methods, with the error bar representing the standard deviation of the mean. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p

Analysis of SINV C:R interaction site mutant growth kinetics and infectivity.

<p><b>A</b>) The one-step growth kinetics of the individual SINV C:R interaction site mutants and parental wild type virus were assessed in HEK293 cells. <b>B</b>) Quantitative analysis of the SINV C:R interaction site mutants and parental wild type virus in regards to the number of infectious units (left y-axis), and the total number of viral particles produced as measured by qRT-PCR (right y-axis). <b>C</b>) The infectivity of the individual SINV C:R interaction site mutants and parental wild type virus as reported as the ratio of total particles per infectious unit as determined using BHK-21 cells. All quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p