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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

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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pntd.0004227.g005: 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.

Mentions: To demonstrate a functional requirement of UBE2A, DDB1 and UBE4B on DENV production in the mosquito, RNAi-mediated gene silencing was performed. As a negative control, gene silencing was also performed for UBE2M (AAEL009026), which was not detected in our RNAseq analysis. Efficacy of RNAi-mediated knockdown was assessed by gene-specific qRT-PCR (S3A Fig). Compared to the infected blood fed dsRNA control, knockdown of UBE2A and DDB1 in infected midguts resulted in a significant reduction of infectious DENV2 titers (Fig 5A) but not viral genomic RNA (Fig 5B). Correspondingly, ratio of PFU to RNA copy number per midgut decreased significantly (Fig 5C). No significant differences were observed for UBE4B or UBE2M knockdown (S3B and S3C Fig).


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)

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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getmorefigures.php?uid=PMC4643912&req=5

pntd.0004227.g005: 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.
Mentions: To demonstrate a functional requirement of UBE2A, DDB1 and UBE4B on DENV production in the mosquito, RNAi-mediated gene silencing was performed. As a negative control, gene silencing was also performed for UBE2M (AAEL009026), which was not detected in our RNAseq analysis. Efficacy of RNAi-mediated knockdown was assessed by gene-specific qRT-PCR (S3A Fig). Compared to the infected blood fed dsRNA control, knockdown of UBE2A and DDB1 in infected midguts resulted in a significant reduction of infectious DENV2 titers (Fig 5A) but not viral genomic RNA (Fig 5B). Correspondingly, ratio of PFU to RNA copy number per midgut decreased significantly (Fig 5C). No significant differences were observed for UBE4B or UBE2M knockdown (S3B and S3C Fig).

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