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Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

Paradkar PN, Duchemin JB, Rodriguez-Andres J, Trinidad L, Walker PJ - PLoS Pathog. (2015)

Bottom Line: It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process.In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes.Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

ABSTRACT
Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

No MeSH data available.


Related in: MedlinePlus

Culex cullin (Cul4) is pro-viral during mosquito infection.Female Culex annulirostris mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV (NY99-4132 strain) for 1 h. At 12 days post-infection, mosquitoes were dissected and total RNA from the midgut and carcass was collected. A. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito carcasses. Control (GFP dsRNA) N = 24; Cul4 dsRNA N = 20. Inset shows individual mosquito data. B. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito carcasses. C. Real-time RT-qPCR was performed using Culex Vir1 primers on mosquito carcasses. D. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito midguts. E. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito midguts. For all real-time RT-qPCR experiments, RpL32 primers were used as an internal control. For plotting, the mean for controls was arbitrarily set at 1 (median shown) and fold-change was calculated for each sample (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). F. Viral titer estimation conducted by plaque assay in mosquito saliva mixed with cell culture medium. Viral titers from individual saliva samples were plotted on the graph (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). Control (GFP dsRNA) N = 17; Cul4 dsRNA N = 17.
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ppat.1005143.g006: Culex cullin (Cul4) is pro-viral during mosquito infection.Female Culex annulirostris mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV (NY99-4132 strain) for 1 h. At 12 days post-infection, mosquitoes were dissected and total RNA from the midgut and carcass was collected. A. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito carcasses. Control (GFP dsRNA) N = 24; Cul4 dsRNA N = 20. Inset shows individual mosquito data. B. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito carcasses. C. Real-time RT-qPCR was performed using Culex Vir1 primers on mosquito carcasses. D. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito midguts. E. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito midguts. For all real-time RT-qPCR experiments, RpL32 primers were used as an internal control. For plotting, the mean for controls was arbitrarily set at 1 (median shown) and fold-change was calculated for each sample (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). F. Viral titer estimation conducted by plaque assay in mosquito saliva mixed with cell culture medium. Viral titers from individual saliva samples were plotted on the graph (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). Control (GFP dsRNA) N = 17; Cul4 dsRNA N = 17.

Mentions: To validate the significance of CxCul4 during viral infection of mosquitoes, female Culex annulirostris mosquitoes were microinjected with dsRNA against CxCul4. As a control, mosquitoes were microinjected with dsRNA against GFP. The mosquitoes were infected with WNV (NY99 strain) by blood-feeding 24 h post-microinjection. At 10 days post-infection, saliva from individual mosquitoes was collected in a capillary tube (see methods) and total RNA was collected from whole mosquito carcass and midgut. As a parallel experiment, mosquitoes were collected at day 2 and day 10 post-infection. Western blot was performed on whole mosquito using anti-CxSTAT and anti-actin antibodies. Results showed increased levels of CxSTAT by day 2 post-infection. The levels decreased to baseline by day 10 post-infection in control mosquitoes (GFP dsRNA); however in mosquitoes with CxCul4 dsRNA, STAT levels remained high (Fig 5E). Real-time RT-qPCR results showed efficient knock-down (>60%) of CxCul4 mRNA in carcass after Cul4 dsRNA microinjections (Fig 6A). The results also showed a decrease (>50%) in viral RNA (NS1) in whole mosquito carcass (Fig 6B) and midgut (Fig 6D) in CxCul4 dsRNA-injected mosquitoes compared with the control, confirming pro-viral effect of CxCul4. Interestingly, real-time RT-qPCR results also showed a significant increase in CxVir1 mRNA expression in carcass (3-fold) (Fig 6C) and midgut (5-fold) (Fig 6E) in CxCul4-knockdown mosquitoes, indicating increased Jak-STAT signaling. Plaque assays performed on mosquito saliva showed a significantly lower virus titer in mosquito saliva microinjected with CxCul4 dsRNA (88 pfu/mosquito) compared with control (GFP dsRNA) (429 pfu/mosquito) (Fig 6F), indicating a lower likelihood of virus transmission by CxCul4 silenced mosquitoes. It should be noted that saliva from 3 mosquitoes with CxCul4 dsRNA injection showed higher viral titers (similar to controls). Further analysis showed that CxCul4 was not efficiently knocked down in these three mosquitoes (Fig 6A, inset). These results further validate that CxCul4 has a pro-viral effect by inhibiting Jak-STAT pathway during WNV infection in mosquitoes. It should also be noted that ~20–30% of mosquitoes from each group did not show any virus in the saliva, possibly indicative of vector competence of these mosquito species.


Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

Paradkar PN, Duchemin JB, Rodriguez-Andres J, Trinidad L, Walker PJ - PLoS Pathog. (2015)

Culex cullin (Cul4) is pro-viral during mosquito infection.Female Culex annulirostris mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV (NY99-4132 strain) for 1 h. At 12 days post-infection, mosquitoes were dissected and total RNA from the midgut and carcass was collected. A. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito carcasses. Control (GFP dsRNA) N = 24; Cul4 dsRNA N = 20. Inset shows individual mosquito data. B. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito carcasses. C. Real-time RT-qPCR was performed using Culex Vir1 primers on mosquito carcasses. D. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito midguts. E. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito midguts. For all real-time RT-qPCR experiments, RpL32 primers were used as an internal control. For plotting, the mean for controls was arbitrarily set at 1 (median shown) and fold-change was calculated for each sample (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). F. Viral titer estimation conducted by plaque assay in mosquito saliva mixed with cell culture medium. Viral titers from individual saliva samples were plotted on the graph (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). Control (GFP dsRNA) N = 17; Cul4 dsRNA N = 17.
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ppat.1005143.g006: Culex cullin (Cul4) is pro-viral during mosquito infection.Female Culex annulirostris mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV (NY99-4132 strain) for 1 h. At 12 days post-infection, mosquitoes were dissected and total RNA from the midgut and carcass was collected. A. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito carcasses. Control (GFP dsRNA) N = 24; Cul4 dsRNA N = 20. Inset shows individual mosquito data. B. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito carcasses. C. Real-time RT-qPCR was performed using Culex Vir1 primers on mosquito carcasses. D. Real-time RT-qPCR was performed using Culex Cul4 primers on mosquito midguts. E. Real-time RT-qPCR was performed using WNV NS1 primers on mosquito midguts. For all real-time RT-qPCR experiments, RpL32 primers were used as an internal control. For plotting, the mean for controls was arbitrarily set at 1 (median shown) and fold-change was calculated for each sample (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). F. Viral titer estimation conducted by plaque assay in mosquito saliva mixed with cell culture medium. Viral titers from individual saliva samples were plotted on the graph (Student’s t-test *p < 0.005, comparing between control and Cul4-knock-down mosquitoes). Control (GFP dsRNA) N = 17; Cul4 dsRNA N = 17.
Mentions: To validate the significance of CxCul4 during viral infection of mosquitoes, female Culex annulirostris mosquitoes were microinjected with dsRNA against CxCul4. As a control, mosquitoes were microinjected with dsRNA against GFP. The mosquitoes were infected with WNV (NY99 strain) by blood-feeding 24 h post-microinjection. At 10 days post-infection, saliva from individual mosquitoes was collected in a capillary tube (see methods) and total RNA was collected from whole mosquito carcass and midgut. As a parallel experiment, mosquitoes were collected at day 2 and day 10 post-infection. Western blot was performed on whole mosquito using anti-CxSTAT and anti-actin antibodies. Results showed increased levels of CxSTAT by day 2 post-infection. The levels decreased to baseline by day 10 post-infection in control mosquitoes (GFP dsRNA); however in mosquitoes with CxCul4 dsRNA, STAT levels remained high (Fig 5E). Real-time RT-qPCR results showed efficient knock-down (>60%) of CxCul4 mRNA in carcass after Cul4 dsRNA microinjections (Fig 6A). The results also showed a decrease (>50%) in viral RNA (NS1) in whole mosquito carcass (Fig 6B) and midgut (Fig 6D) in CxCul4 dsRNA-injected mosquitoes compared with the control, confirming pro-viral effect of CxCul4. Interestingly, real-time RT-qPCR results also showed a significant increase in CxVir1 mRNA expression in carcass (3-fold) (Fig 6C) and midgut (5-fold) (Fig 6E) in CxCul4-knockdown mosquitoes, indicating increased Jak-STAT signaling. Plaque assays performed on mosquito saliva showed a significantly lower virus titer in mosquito saliva microinjected with CxCul4 dsRNA (88 pfu/mosquito) compared with control (GFP dsRNA) (429 pfu/mosquito) (Fig 6F), indicating a lower likelihood of virus transmission by CxCul4 silenced mosquitoes. It should be noted that saliva from 3 mosquitoes with CxCul4 dsRNA injection showed higher viral titers (similar to controls). Further analysis showed that CxCul4 was not efficiently knocked down in these three mosquitoes (Fig 6A, inset). These results further validate that CxCul4 has a pro-viral effect by inhibiting Jak-STAT pathway during WNV infection in mosquitoes. It should also be noted that ~20–30% of mosquitoes from each group did not show any virus in the saliva, possibly indicative of vector competence of these mosquito species.

Bottom Line: It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process.In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes.Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

ABSTRACT
Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

No MeSH data available.


Related in: MedlinePlus