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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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Proteasome inhibition decouples infectious DENV2 production from viral RNA replication in THP-1 cells.(A) No cytotoxicity was observed in THP-1 cells in the presence of β-lactone and genistein for 48 h using a cell viability assay. Mean ± SD. N = 4. (B) Using flow cytometry, β-lactone does not block uptake of DENV2 immune complexes. Genistein, an inhibitor of Fc receptor-mediated entry blocked uptake. (C) Alexa Fluor 647 labeled DENV2 opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hpi. (D) Proteasome inhibition showed a dose-dependent decrease in plaque titers 48 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01, ***p<0.001. (E) DENV2 RNA copy number per GAPDH showed no significant difference between β-lactone treated and DMSO control 48 hpi. (F) Ratio of infectious DENV2 to genomic copies showed a dose-dependent decrease after β-lactone treatment. Mean ± SD. N = 4. Student’s t test, *p < 0.05, **p<0.01.
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pntd.0004058.g001: Proteasome inhibition decouples infectious DENV2 production from viral RNA replication in THP-1 cells.(A) No cytotoxicity was observed in THP-1 cells in the presence of β-lactone and genistein for 48 h using a cell viability assay. Mean ± SD. N = 4. (B) Using flow cytometry, β-lactone does not block uptake of DENV2 immune complexes. Genistein, an inhibitor of Fc receptor-mediated entry blocked uptake. (C) Alexa Fluor 647 labeled DENV2 opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hpi. (D) Proteasome inhibition showed a dose-dependent decrease in plaque titers 48 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01, ***p<0.001. (E) DENV2 RNA copy number per GAPDH showed no significant difference between β-lactone treated and DMSO control 48 hpi. (F) Ratio of infectious DENV2 to genomic copies showed a dose-dependent decrease after β-lactone treatment. Mean ± SD. N = 4. Student’s t test, *p < 0.05, **p<0.01.

Mentions: To demonstrate that proteasome inhibition with clasto lactacystin β-lactone (β-lactone), a widely used proteasome inhibitor, indeed did not alter virus entry at non-toxic levels (Fig 1A), we measured DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt)-labeled DENV in β-lactone treated cells. DENV2 opsonized with enhancing levels of humanized 3H5 monoclonal antibody (h3H5) was added to a THP-1 subclone with increased susceptibility to antibody-dependent infection [21]. Cells were pretreated with β-lactone or DMSO as a vehicle control. Results showed that the proportion of DiD-positive cells were similar to that of DMSO control (Fig 1B). As a positive control, we treated cells with genistein, a specific tyrosine kinase inhibitor that blocks Fc receptor-mediated phagocytosis [22,23]. Genistein treatment significantly reduced uptake of h3H5-opsonized DENV in a concentration-dependent manner (Fig 1B) [24]. Similar observations were made with confocal microscopy where Alexa Fluor 647 labeled DENV [25] opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hours post infection (hpi) (Fig 1C). Altogether, these data indicate that any antiviral effect observed during β-lactone treatment is independent of DENV entry.


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)

Proteasome inhibition decouples infectious DENV2 production from viral RNA replication in THP-1 cells.(A) No cytotoxicity was observed in THP-1 cells in the presence of β-lactone and genistein for 48 h using a cell viability assay. Mean ± SD. N = 4. (B) Using flow cytometry, β-lactone does not block uptake of DENV2 immune complexes. Genistein, an inhibitor of Fc receptor-mediated entry blocked uptake. (C) Alexa Fluor 647 labeled DENV2 opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hpi. (D) Proteasome inhibition showed a dose-dependent decrease in plaque titers 48 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01, ***p<0.001. (E) DENV2 RNA copy number per GAPDH showed no significant difference between β-lactone treated and DMSO control 48 hpi. (F) Ratio of infectious DENV2 to genomic copies showed a dose-dependent decrease after β-lactone treatment. Mean ± SD. N = 4. Student’s t test, *p < 0.05, **p<0.01.
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pntd.0004058.g001: Proteasome inhibition decouples infectious DENV2 production from viral RNA replication in THP-1 cells.(A) No cytotoxicity was observed in THP-1 cells in the presence of β-lactone and genistein for 48 h using a cell viability assay. Mean ± SD. N = 4. (B) Using flow cytometry, β-lactone does not block uptake of DENV2 immune complexes. Genistein, an inhibitor of Fc receptor-mediated entry blocked uptake. (C) Alexa Fluor 647 labeled DENV2 opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hpi. (D) Proteasome inhibition showed a dose-dependent decrease in plaque titers 48 hpi. Mean ± SD. N = 4. Student’s t test, **p<0.01, ***p<0.001. (E) DENV2 RNA copy number per GAPDH showed no significant difference between β-lactone treated and DMSO control 48 hpi. (F) Ratio of infectious DENV2 to genomic copies showed a dose-dependent decrease after β-lactone treatment. Mean ± SD. N = 4. Student’s t test, *p < 0.05, **p<0.01.
Mentions: To demonstrate that proteasome inhibition with clasto lactacystin β-lactone (β-lactone), a widely used proteasome inhibitor, indeed did not alter virus entry at non-toxic levels (Fig 1A), we measured DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt)-labeled DENV in β-lactone treated cells. DENV2 opsonized with enhancing levels of humanized 3H5 monoclonal antibody (h3H5) was added to a THP-1 subclone with increased susceptibility to antibody-dependent infection [21]. Cells were pretreated with β-lactone or DMSO as a vehicle control. Results showed that the proportion of DiD-positive cells were similar to that of DMSO control (Fig 1B). As a positive control, we treated cells with genistein, a specific tyrosine kinase inhibitor that blocks Fc receptor-mediated phagocytosis [22,23]. Genistein treatment significantly reduced uptake of h3H5-opsonized DENV in a concentration-dependent manner (Fig 1B) [24]. Similar observations were made with confocal microscopy where Alexa Fluor 647 labeled DENV [25] opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at 2 hours post infection (hpi) (Fig 1C). Altogether, these data indicate that any antiviral effect observed during β-lactone treatment is independent of DENV entry.

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