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. 2015 Nov 13;9(11):e0004227.
doi: 10.1371/journal.pntd.0004227. eCollection 2015 Nov.

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Milly M Choy  1 , October M Sessions  1 , Duane J Gubler  1 , Eng Eong Ooi  1   2
Affiliations

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Milly M Choy et al. PLoS Negl Trop Dis. .

Abstract

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 Aedes aegypti 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 in vivo 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of DENV-2 replication in the midguts and heads/thoraces of Ae. aegypti following ingestion of an infectious blood meal.
(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).
Fig 2
Fig 2. Proteasome inhibition of β2 and β5 subunits decouples infectious DENV-2 production from viral RNA replication in mosquito midguts.
(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.
Fig 3
Fig 3. Organ- and time-specific regulation of genes belonging to the UPP in response to DENV infection.
(A) Experimental workflow of transcriptome analysis of Ae. aegypti midgut 8 dpbm. (B) Validation of differentially regulated UPP-specific genes using qRT-PCR. Expression levels of UPP genes in midgut 8 dpbm and head/thorax 21 dpbm were compared. Contrasting expression levels of UBE2A, DDB1 and UBE4B were observed, with these genes being down-regulated in the midgut, but up-regulated in the head/thorax when compared with uninfected midguts or heads/thoraces respectively. Mean ± SEM, N = 12. Student’s t test, *p < 0.05. (C) Gene expression levels in individual infected midguts were measured using qRT-PCR, normalized to GAPDH and compared to midguts from uninfected blood fed mosquitoes. Mean ± SEM. N = 12–16. Student’s t test, *p < 0.05.
Fig 4
Fig 4. Ingestion of blood meal does not modulate gene expression of UBE2A and DDB1.
Sugar fed and blood fed mosquitoes were harvested at different time-points and individual midguts were dissected for analyses. Gene expression levels for (A) UBE2A and (B) DDB1 were measured using qRT-PCR and normalized to GAPDH. Expression levels of UBE2A and DDB1 in blood fed mosquitoes remained consistently unchanged over the course of 14 days, whereas ingestion of a sugar meal increases expression levels of UBE2A and DDB1 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.
Fig 5
Fig 5. Knockdown of UBE2A and DDB1 decouples infectious DENV2 production from viral RNA replication in mosquitoes.
(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.
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This work was supported by the Singapore National Research Foundation under its Clinician-Scientist Award (NMRC/CSA/0060/2014), administered by the National Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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