Production of Infectious Dengue Virus in <i>Aedes aegypti</i> Is Dependent on the Ubiquitin Proteasome Pathway

<div><p>Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of <i>Aedes aegypti</i> and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show <i>in vivo</i> that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.</p></div

Proteasome inhibition of β2 and β5 subunits decouples infectious DENV-2 production from viral RNA replication in mosquito midguts.

<p>(A) Silencing efficiencies of β subunits of the proteasome were determined by gene specific qPCR, and expression values were normalized against dsRNA control targeting random sequences from pGEM T easy vector 10 days after dsRNA inoculation. N = 10. (B) No statistically significant difference was observed in virus titer per midgut at 6 dpbm after knockdown of β1, β2 and β5 subunits. Mean ± SEM, N = 7–16. (C) No statistically significant differences were observed in DENV2 viral RNA levels per midgut 6 dpbm after β1, β2 and β5 subunits knockdown. Mean ± SEM, N = 7–16. (D) log(PFU/Copy Number) was significantly lower after β2 and β5 knockdown. Mean ± SEM, N = 20–22. Student’s t test, **p<0.01.</p

Knockdown of UBE2A and DDB1 decouples infectious DENV2 production from viral RNA replication in mosquitoes.

<p>(A) In the infected midguts, virus titers declined significantly after knockdown of UBE2A and DDB1 at 6 dpbm. N = 12–16. Student’s t test, *p < 0.05. (B) In the infected midguts, no statistically significant differences were observed in DENV2 viral RNA levels 6 dpbm after gene knockdown. N = 12–16. (C) Ratio of midgut infectious titers to viral RNA levels 6 dpbm after gene knockdown. N = 12–16. Student’s t test, *p < 0.05. (D) In infected heads/thoraces (HT), virus titers at 8 days post intra-thoracic inoculation declined significantly after knockdown of UBE2A and DDB1. N = 8–10. Student’s t test, **p < 0.01, ***p<0.001. (E) In infected heads/thoraces (HT), no statistically significant differences were observed in DENV2 viral RNA levels after gene knockdown. N = 8–10. (F) Ratio of head/thorax (HT) infectious titers to viral RNA levels after gene knockdown 8 dpi. N = 12–16. Student’s t test, **p<0.01.</p

Characterization of DENV-2 replication in the midguts and heads/thoraces of <i>Ae</i>. <i>aegypti</i> following ingestion of an infectious blood meal.

<p>(A) In the midgut, viral titers increased linearly until 8 dpbm and declined thereafter. In contrast, viral RNA remained stable between 8 to 21 dpbm. Mean ± SEM, N = 8–10. (B) In the heads/thoraces (HT), the increase in both infectious particles and viral RNA are positively correlated over time. Viral RNA copy number increases with increasing viral titers. Mean ± SEM, N = 8–10. (C-D) A corresponding decrease in PFU/Copy number was observed in the midgut over time, with no significant change in the head/thorax (HT).</p

Ingestion of blood meal does not modulate gene expression of <i>UBE2A</i> and <i>DDB1</i>.

<p>Sugar fed and blood fed mosquitoes were harvested at different time-points and individual midguts were dissected for analyses. Gene expression levels for (A) <i>UBE2A</i> and (B) <i>DDB1</i> were measured using qRT-PCR and normalized to GAPDH. Expression levels of <i>UBE2A</i> and <i>DDB1</i> in blood fed mosquitoes remained consistently unchanged over the course of 14 days, whereas ingestion of a sugar meal increases expression levels of <i>UBE2A</i> and <i>DDB1</i> significantly until 8 days relative to the blood fed mosquitoes. Mean ± SEM. N = 12. Student’s t test, **p < 0.01, ***p<0.001, ****p<0.0001.</p

Percentage of DENV2-infected mosquitoes after knockdown of proteasome subunits (p value; Fischer’s Exact Test).

<p>Percentage of DENV2-infected mosquitoes after knockdown of proteasome subunits (p value; Fischer’s Exact Test).</p

Cross-platform analysis of DENV host factors.

<p>The number of host factors identified in each of the RNAi screens listed compared to the differentially regulated isoforms and differentially spliced genes for both the wild-type DENV1-16007 and vaccine strain DENV1-PDK13. Percentages of overlap are indicated in parenthesis. The unpublished DENV screen by Jamison SF, Garcia-Blanco, M.A. utilize the same siRNA library, cell line, general methodology and statistical analysis described in Le Sommer et al 2012. The unpublished hits from the WNV screen are the full, unannotated results from the published Krishnan et al 2008 study.</p

Cross-platform analysis of human-Flaviviridae interactions.

<p>Members of eleven canonical pathways that have been previously implicated to be important for DENV propagation were interrogated for their appearance in an RNAi screen for Flaviviridae host factors, direct interaction with DENV gene products, differential regulation in a microarray study, differential isoform regulation following infection with DENV1-16007 and/or differential isoform regulation following infection with DENV1-PDK13. Host factors identified in published and/or unpublished Flaviviridae RNAi screens <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Krishnan1" target="_blank">[10]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Sessions1" target="_blank">[11]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Li1" target="_blank">[48]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Tai1" target="_blank">[49]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-LeSommer1" target="_blank">[51]</a> (Jamison and Garcia-Blanco, unpublished data) were combined and are indicated by a black bar. Host proteins found to interact DENV gene products <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-LeBreton1" target="_blank">[47]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Ward1" target="_blank">[50]</a> were combined and are indicated by a grey bar. Host genes found to be differentially regulated in microarray studies <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Balas1" target="_blank">[12]</a>–<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Warke1" target="_blank">[29]</a> are indicated by the striped bar. A red bar indicates host isoforms found to be differentially regulated during infection with DENV1-16007. A blue bar indicates host isoforms found to be differentially regulated during infection with DENV1-PDK13.</p

Differential regulation of transcripts in response to DENV1 infection.

<p>Number of events in each category (Rows) predicted by Cuffdiff <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Trapnell1" target="_blank">[40]</a> (Top) and MISO <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002107#pntd.0002107-Katz1" target="_blank">[41]</a> (Bottom) for Uninfected cells vs. DENV1-16007 infected cells (First column), Uninfected cells vs. DENV1-PDK13 infected cells (Second column). The number of up- and down-regulated events is also described for the gene and isoform categories. Superscript “a” indicates biological triplicates while superscript “b” indicates single measurement of the sample. Abbreviations used: differentially expressed genes (Genes exp), differentially expressed isoforms (Isoforms exp), differentially expressed transcriptional start site group (TSS group exp), differentially expressed coding sequence (CDS exp), differential coding output (CDS), differential promoter use (Promoters), differential splicing (Splicing), skipped exon (SE), mutually exclusive exons (MXE), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), alternative first exon (AFE), retained intron (RI) and tandem untranslated region (Tandem UTR).</p

Experimental design and analysis of infection.

<p><b>A</b>. Timeline of experimental procedure and data analysis steps. <b>B</b>. Quantification of viral copy number normalized to the cellular B2M gene expression at 0, 6, 12, 20 and 30 hours post infection. Asterisks indicate a p-value of less than or equal to 0.05. <b>C</b>. Immunofluorescence analysis of Huh7 cells either mock-infected (top panel), infected with DENV1-16007 (middle panel) or DENV1- PDK13 (bottom panel) and probed with antibody against prM protein (2H2) followed by FITC-conjugated goat anti-mouse IgG at 20 h post-infection. The cell nuclei were counter-stained with Evan's Blue.</p