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

Choy MM, Sessions OM, Gubler DJ, Ooi EE - PLoS Negl Trop Dis (2015)

Bottom Line: 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.Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.

View Article: PubMed Central - PubMed

Affiliation: Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore.

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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Related in: MedlinePlus

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).
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pntd.0004227.g001: 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).

Mentions: We first tested if the observation described previously [22] could be replicated in our hands by characterizing virus replication in the midgut of Ae. aegypti following ingestion of blood spiked with DENV2. DENV2 infection was detected in midgut epithelial cells as early as 2 dpbm, peaked at 8 dpbm and decreased thereafter (Fig 1A). The amount of viral antigen detected using immunofluorescence decreased starting at 10 dpbm as well (S1 Fig). However, measurement of DENV2 RNA using qRT-PCR revealed no significant reduction in the viral RNA copy number as late as 21 dpbm (Fig 1A). In contrast, both DENV2 titers and viral RNA copy number in the head/thorax were positively correlated through to the limit of the lifespan of Ae. aegypti in the laboratory (Fig 1B). Correspondingly, the ratio of PFU to RNA copy number decreased significantly over time in the midgut but not in the head/thorax (Fig 1C and 1D). This observation recapitulated previously reported findings [22] and guides our experimental design to study proteasome function 8 days post infection (dpi).


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

Choy MM, Sessions OM, Gubler DJ, Ooi EE - PLoS Negl Trop Dis (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643912&req=5

pntd.0004227.g001: 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).
Mentions: We first tested if the observation described previously [22] could be replicated in our hands by characterizing virus replication in the midgut of Ae. aegypti following ingestion of blood spiked with DENV2. DENV2 infection was detected in midgut epithelial cells as early as 2 dpbm, peaked at 8 dpbm and decreased thereafter (Fig 1A). The amount of viral antigen detected using immunofluorescence decreased starting at 10 dpbm as well (S1 Fig). However, measurement of DENV2 RNA using qRT-PCR revealed no significant reduction in the viral RNA copy number as late as 21 dpbm (Fig 1A). In contrast, both DENV2 titers and viral RNA copy number in the head/thorax were positively correlated through to the limit of the lifespan of Ae. aegypti in the laboratory (Fig 1B). Correspondingly, the ratio of PFU to RNA copy number decreased significantly over time in the midgut but not in the head/thorax (Fig 1C and 1D). This observation recapitulated previously reported findings [22] and guides our experimental design to study proteasome function 8 days post infection (dpi).

Bottom Line: 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.Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.

View Article: PubMed Central - PubMed

Affiliation: Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore.

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.

Show MeSH
Related in: MedlinePlus