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Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

Choy MM, Zhang SL, Costa VV, Tan HC, Horrevorts S, Ooi EE - PLoS Negl Trop Dis (2015)

Bottom Line: Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined.Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress.Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes.

View Article: PubMed Central - PubMed

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

ABSTRACT
The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

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Related in: MedlinePlus

DENV2 egress is dependent on proteasome function.(A) Ratio of viral genomic RNA from cell culture supernatant to infectious DENV2 showed no significant differences after β-lactone treatment at different drug concentrations, compared to DMSO control, suggesting that the treatment with β-lactone did not result in reduced DENV2 maturation. Mean ± SD. N = 4. (B) A dose-dependent accumulation of E protein in cells is observed with no difference in the levels of NS3 at 24 hpi. (C) Confocal analysis showed accumulation of structural proteins (prM and E, in red) in β-lactone treated compared to DMSO treated cells at 24 hpi with strong co-localization with Golgi (in green), suggesting that egress of viral particles were impaired in presence of UPP inhibition (Scale bar = 10 μm). (D) Accumulation of viral particles in intra-cytoplasmic vacuoles (50 nm) in β-lactone compared to DMSO treated cells was observed using electron microscopy, indicating that DENV2 RNA could replicate and be packaged with structural proteins (Scale bar = 500 nm). (E) Ratio of RNase to non-RNase treated cells after β-lactone treatment is significantly higher compared to the DMSO control 24 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01.
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pntd.0004058.g002: DENV2 egress is dependent on proteasome function.(A) Ratio of viral genomic RNA from cell culture supernatant to infectious DENV2 showed no significant differences after β-lactone treatment at different drug concentrations, compared to DMSO control, suggesting that the treatment with β-lactone did not result in reduced DENV2 maturation. Mean ± SD. N = 4. (B) A dose-dependent accumulation of E protein in cells is observed with no difference in the levels of NS3 at 24 hpi. (C) Confocal analysis showed accumulation of structural proteins (prM and E, in red) in β-lactone treated compared to DMSO treated cells at 24 hpi with strong co-localization with Golgi (in green), suggesting that egress of viral particles were impaired in presence of UPP inhibition (Scale bar = 10 μm). (D) Accumulation of viral particles in intra-cytoplasmic vacuoles (50 nm) in β-lactone compared to DMSO treated cells was observed using electron microscopy, indicating that DENV2 RNA could replicate and be packaged with structural proteins (Scale bar = 500 nm). (E) Ratio of RNase to non-RNase treated cells after β-lactone treatment is significantly higher compared to the DMSO control 24 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01.

Mentions: The observed decoupling of infectious DENV production from RNA replication in β-lactone treated cells has three possible explanations. Firstly, newly formed DENV were released from the cells, but as immature and non-infectious particles. Alternatively, normal proteasome function may be critical for either virus assembly or egress from infected cells. To test the first possibility, we measured viral RNA in the cell culture supernatant by qRT-PCR and compared it with the amount of infectious virions [26]. Ratio of viral genomic RNA to PFU showed no statistically significant difference regardless of the drug concentration used (Fig 2A), indicating that treatment with β-lactone did not result in the release of proportionately more immature DENV compared to DMSO treated cells.


Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

Choy MM, Zhang SL, Costa VV, Tan HC, Horrevorts S, Ooi EE - PLoS Negl Trop Dis (2015)

DENV2 egress is dependent on proteasome function.(A) Ratio of viral genomic RNA from cell culture supernatant to infectious DENV2 showed no significant differences after β-lactone treatment at different drug concentrations, compared to DMSO control, suggesting that the treatment with β-lactone did not result in reduced DENV2 maturation. Mean ± SD. N = 4. (B) A dose-dependent accumulation of E protein in cells is observed with no difference in the levels of NS3 at 24 hpi. (C) Confocal analysis showed accumulation of structural proteins (prM and E, in red) in β-lactone treated compared to DMSO treated cells at 24 hpi with strong co-localization with Golgi (in green), suggesting that egress of viral particles were impaired in presence of UPP inhibition (Scale bar = 10 μm). (D) Accumulation of viral particles in intra-cytoplasmic vacuoles (50 nm) in β-lactone compared to DMSO treated cells was observed using electron microscopy, indicating that DENV2 RNA could replicate and be packaged with structural proteins (Scale bar = 500 nm). (E) Ratio of RNase to non-RNase treated cells after β-lactone treatment is significantly higher compared to the DMSO control 24 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643959&req=5

pntd.0004058.g002: DENV2 egress is dependent on proteasome function.(A) Ratio of viral genomic RNA from cell culture supernatant to infectious DENV2 showed no significant differences after β-lactone treatment at different drug concentrations, compared to DMSO control, suggesting that the treatment with β-lactone did not result in reduced DENV2 maturation. Mean ± SD. N = 4. (B) A dose-dependent accumulation of E protein in cells is observed with no difference in the levels of NS3 at 24 hpi. (C) Confocal analysis showed accumulation of structural proteins (prM and E, in red) in β-lactone treated compared to DMSO treated cells at 24 hpi with strong co-localization with Golgi (in green), suggesting that egress of viral particles were impaired in presence of UPP inhibition (Scale bar = 10 μm). (D) Accumulation of viral particles in intra-cytoplasmic vacuoles (50 nm) in β-lactone compared to DMSO treated cells was observed using electron microscopy, indicating that DENV2 RNA could replicate and be packaged with structural proteins (Scale bar = 500 nm). (E) Ratio of RNase to non-RNase treated cells after β-lactone treatment is significantly higher compared to the DMSO control 24 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01.
Mentions: The observed decoupling of infectious DENV production from RNA replication in β-lactone treated cells has three possible explanations. Firstly, newly formed DENV were released from the cells, but as immature and non-infectious particles. Alternatively, normal proteasome function may be critical for either virus assembly or egress from infected cells. To test the first possibility, we measured viral RNA in the cell culture supernatant by qRT-PCR and compared it with the amount of infectious virions [26]. Ratio of viral genomic RNA to PFU showed no statistically significant difference regardless of the drug concentration used (Fig 2A), indicating that treatment with β-lactone did not result in the release of proportionately more immature DENV compared to DMSO treated cells.

Bottom Line: Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined.Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress.Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes.

View Article: PubMed Central - PubMed

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

ABSTRACT
The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

Show MeSH
Related in: MedlinePlus