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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

Bortezomib reduced viral load and signs of dengue pathology in C57BL/6 mice.WT mice infected intraperitonealy with DENV2 were treated with bortezomib 6 hpi and analyzed at 24, 48 and 72 hpi for further analysis. (A) For quantification of NS3+ cells, the mean cell count in 20 alternate microscopic high-power fields (x400) was measured. Quantification was done only in the red pulp of spleen. Bortezomib treated mice showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. Mean ± SEM. N = 4–5. Student’s t test, **p<0.01. (B) Representative serial sections from spleen of each group of mice stained with anti-DENV NS3 antibody at 24, 48 and 72 hpi. Multiple sections of each tissue were examined for staining (60x magnification). The inset is a representation of the spleen from mock infected mice without (top panel) or with (bottom panel) bortezomib treatment. The top panel shows DENV-infected spleen without bortezomib treatment 24, 48 and 72 hpi. The bottom panel shows DENV-infected spleen with bortezomib treatment 24, 48 and 72 hpi. Positive staining for NS3 is brown while hematoxylin counterstaining is blue. Bortezomib treated mice also showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. (C) RNA copy number in the spleen was reduced in bortezomib treated mice compared to vehicle control at 48 hpi. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05. (D-E) The vehicle control experienced a drop in platelet count over the first 48 hours of infection before recovering by day 3, and experienced a significant rise in hematocrit values that peaked 24 hpi. On the other hand, no significant changes were observed for the platelet count 24–72 hpi, and hematocrit levels 0–72 hpi in mice after bortezomib treatment, suggesting the efficiency of bortezomib in alleviating disease symptoms. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05, ****p<0.0001. (F-G) DENV2-infected vehicle control displayed an increased systemic level of MCPT1, indicative of mast cell activation, in mouse serum and spleen when compared to bortezomib treated mice at all time-points. (H) Levels of TNF-α were decreased in bortezomib treated mice 24 hpi when compared to the vehicle control. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05.
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pntd.0004058.g006: Bortezomib reduced viral load and signs of dengue pathology in C57BL/6 mice.WT mice infected intraperitonealy with DENV2 were treated with bortezomib 6 hpi and analyzed at 24, 48 and 72 hpi for further analysis. (A) For quantification of NS3+ cells, the mean cell count in 20 alternate microscopic high-power fields (x400) was measured. Quantification was done only in the red pulp of spleen. Bortezomib treated mice showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. Mean ± SEM. N = 4–5. Student’s t test, **p<0.01. (B) Representative serial sections from spleen of each group of mice stained with anti-DENV NS3 antibody at 24, 48 and 72 hpi. Multiple sections of each tissue were examined for staining (60x magnification). The inset is a representation of the spleen from mock infected mice without (top panel) or with (bottom panel) bortezomib treatment. The top panel shows DENV-infected spleen without bortezomib treatment 24, 48 and 72 hpi. The bottom panel shows DENV-infected spleen with bortezomib treatment 24, 48 and 72 hpi. Positive staining for NS3 is brown while hematoxylin counterstaining is blue. Bortezomib treated mice also showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. (C) RNA copy number in the spleen was reduced in bortezomib treated mice compared to vehicle control at 48 hpi. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05. (D-E) The vehicle control experienced a drop in platelet count over the first 48 hours of infection before recovering by day 3, and experienced a significant rise in hematocrit values that peaked 24 hpi. On the other hand, no significant changes were observed for the platelet count 24–72 hpi, and hematocrit levels 0–72 hpi in mice after bortezomib treatment, suggesting the efficiency of bortezomib in alleviating disease symptoms. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05, ****p<0.0001. (F-G) DENV2-infected vehicle control displayed an increased systemic level of MCPT1, indicative of mast cell activation, in mouse serum and spleen when compared to bortezomib treated mice at all time-points. (H) Levels of TNF-α were decreased in bortezomib treated mice 24 hpi when compared to the vehicle control. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05.

Mentions: We treated C57BL/6 mice infected with DENV2 with a single dose of bortezomib at 6 hpi. The dose was based on that licensed for the treatment of multiple myeloma. The spleen was chosen for analysis since previous work has shown that this animal model produces no detectable viremia but the viral load in the spleen correlated with the degree of plasma leakage [32]. Using immunohistochemistry, we observed that mice treated with bortezomib showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control (Fig 6A and 6B). No difference in NS3-positive cells was observed at 72 hpi, which is consistent with previously reported data that this strain of mice clears DENV infection rapidly without intervention [32]. Consistently, a significant difference in viral RNA genome copies was also observed at 48 hpi in bortezomib treated cells (Fig 6C). Both observations suggest that proteasome inhibition could inhibit virus egress and hence spread in mammals. Along with reducing viral burden, bortezomib treatment also reduced the degree of thrombocytopenia (Fig 6D) and plasma leakage (Fig 6E) compared to control animals.


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)

Bortezomib reduced viral load and signs of dengue pathology in C57BL/6 mice.WT mice infected intraperitonealy with DENV2 were treated with bortezomib 6 hpi and analyzed at 24, 48 and 72 hpi for further analysis. (A) For quantification of NS3+ cells, the mean cell count in 20 alternate microscopic high-power fields (x400) was measured. Quantification was done only in the red pulp of spleen. Bortezomib treated mice showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. Mean ± SEM. N = 4–5. Student’s t test, **p<0.01. (B) Representative serial sections from spleen of each group of mice stained with anti-DENV NS3 antibody at 24, 48 and 72 hpi. Multiple sections of each tissue were examined for staining (60x magnification). The inset is a representation of the spleen from mock infected mice without (top panel) or with (bottom panel) bortezomib treatment. The top panel shows DENV-infected spleen without bortezomib treatment 24, 48 and 72 hpi. The bottom panel shows DENV-infected spleen with bortezomib treatment 24, 48 and 72 hpi. Positive staining for NS3 is brown while hematoxylin counterstaining is blue. Bortezomib treated mice also showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. (C) RNA copy number in the spleen was reduced in bortezomib treated mice compared to vehicle control at 48 hpi. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05. (D-E) The vehicle control experienced a drop in platelet count over the first 48 hours of infection before recovering by day 3, and experienced a significant rise in hematocrit values that peaked 24 hpi. On the other hand, no significant changes were observed for the platelet count 24–72 hpi, and hematocrit levels 0–72 hpi in mice after bortezomib treatment, suggesting the efficiency of bortezomib in alleviating disease symptoms. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05, ****p<0.0001. (F-G) DENV2-infected vehicle control displayed an increased systemic level of MCPT1, indicative of mast cell activation, in mouse serum and spleen when compared to bortezomib treated mice at all time-points. (H) Levels of TNF-α were decreased in bortezomib treated mice 24 hpi when compared to the vehicle control. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05.
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pntd.0004058.g006: Bortezomib reduced viral load and signs of dengue pathology in C57BL/6 mice.WT mice infected intraperitonealy with DENV2 were treated with bortezomib 6 hpi and analyzed at 24, 48 and 72 hpi for further analysis. (A) For quantification of NS3+ cells, the mean cell count in 20 alternate microscopic high-power fields (x400) was measured. Quantification was done only in the red pulp of spleen. Bortezomib treated mice showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. Mean ± SEM. N = 4–5. Student’s t test, **p<0.01. (B) Representative serial sections from spleen of each group of mice stained with anti-DENV NS3 antibody at 24, 48 and 72 hpi. Multiple sections of each tissue were examined for staining (60x magnification). The inset is a representation of the spleen from mock infected mice without (top panel) or with (bottom panel) bortezomib treatment. The top panel shows DENV-infected spleen without bortezomib treatment 24, 48 and 72 hpi. The bottom panel shows DENV-infected spleen with bortezomib treatment 24, 48 and 72 hpi. Positive staining for NS3 is brown while hematoxylin counterstaining is blue. Bortezomib treated mice also showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control. (C) RNA copy number in the spleen was reduced in bortezomib treated mice compared to vehicle control at 48 hpi. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05. (D-E) The vehicle control experienced a drop in platelet count over the first 48 hours of infection before recovering by day 3, and experienced a significant rise in hematocrit values that peaked 24 hpi. On the other hand, no significant changes were observed for the platelet count 24–72 hpi, and hematocrit levels 0–72 hpi in mice after bortezomib treatment, suggesting the efficiency of bortezomib in alleviating disease symptoms. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05, ****p<0.0001. (F-G) DENV2-infected vehicle control displayed an increased systemic level of MCPT1, indicative of mast cell activation, in mouse serum and spleen when compared to bortezomib treated mice at all time-points. (H) Levels of TNF-α were decreased in bortezomib treated mice 24 hpi when compared to the vehicle control. Mean ± SEM. N = 4–5. Student’s t test, *p<0.05.
Mentions: We treated C57BL/6 mice infected with DENV2 with a single dose of bortezomib at 6 hpi. The dose was based on that licensed for the treatment of multiple myeloma. The spleen was chosen for analysis since previous work has shown that this animal model produces no detectable viremia but the viral load in the spleen correlated with the degree of plasma leakage [32]. Using immunohistochemistry, we observed that mice treated with bortezomib showed significantly reduced number of DENV infected cells in the red pulp of the spleen at 24 and 48 hpi compared to vehicle control (Fig 6A and 6B). No difference in NS3-positive cells was observed at 72 hpi, which is consistent with previously reported data that this strain of mice clears DENV infection rapidly without intervention [32]. Consistently, a significant difference in viral RNA genome copies was also observed at 48 hpi in bortezomib treated cells (Fig 6C). Both observations suggest that proteasome inhibition could inhibit virus egress and hence spread in mammals. Along with reducing viral burden, bortezomib treatment also reduced the degree of thrombocytopenia (Fig 6D) and plasma leakage (Fig 6E) compared to control animals.

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