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Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions.

Cime-Castillo J, Delannoy P, Mendoza-Hernández G, Monroy-Martínez V, Harduin-Lepers A, Lanz-Mendoza H, Hernández-Hernández Fde L, Zenteno E, Cabello-Gutiérrez C, Ruiz-Ordaz BH - Biomed Res Int (2015)

Bottom Line: AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface.Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome.Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology and Biotechnology Department, Biomedical Research Institute, National University of México (UNAM), 04510 México City, Mexico.

ABSTRACT
Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

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DENV-mammalian cells internalization assay. (a) DENV internalization by LLC-MK2 and CHO cells. The plot shows the internalization of [35S]-methionine-radiolabeled DENV by LLC-MK2 and CHO cells in the absence (lanes 2 and 6) and presence (lanes 3 and 7) of Ae. aegypti SG protein extract (SGH) and in the presence of SGH pretreated with sialidase before DENV incubation (lanes 4 and 8). DENV was mixed with SGH (from 80 SGs), which was pretreated or untreated with sialidase, before infecting mammalian cells with the DENV-SGH mixture. In the plot, the y-axis shows the counts per min of internalized DENV, *P < 0.05. (b) DENV internalization by CHO cells in the presence of different amounts of SGH. The plot shows that DENV internalization was enhanced by the presence of the protein extract from five SGs, which was dose dependent.
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fig6: DENV-mammalian cells internalization assay. (a) DENV internalization by LLC-MK2 and CHO cells. The plot shows the internalization of [35S]-methionine-radiolabeled DENV by LLC-MK2 and CHO cells in the absence (lanes 2 and 6) and presence (lanes 3 and 7) of Ae. aegypti SG protein extract (SGH) and in the presence of SGH pretreated with sialidase before DENV incubation (lanes 4 and 8). DENV was mixed with SGH (from 80 SGs), which was pretreated or untreated with sialidase, before infecting mammalian cells with the DENV-SGH mixture. In the plot, the y-axis shows the counts per min of internalized DENV, *P < 0.05. (b) DENV internalization by CHO cells in the presence of different amounts of SGH. The plot shows that DENV internalization was enhanced by the presence of the protein extract from five SGs, which was dose dependent.

Mentions: It is known that Ae. aegypti saliva enhances West Nile and Cache Valley virus infections, but it is unknown whether Aedes saliva can modulate DENV infections [6]. Based on our detection of interactions between DENV and salivary glycoproteins, we evaluated the possible participation of the Ae. aegypti SG protein extract in the modulation of DENV infection in different mammalian cell lines (LLCMK2 and CHO WT) using a DENV internalization assay, in the presence or absence of SG extracts. We found that DENV infection was enhanced in the presence of SG extract in both mammalian cell lines (Figure 6(a)). CHO cells appeared to be more permissive (fourfold enhancement; Figure 6(a), lane 7) than LLCMK2 (twofold enhancement; Figure 6(a), lane 3). We pretreated the SG protein extract with sialidase before the internalization assay to evaluate the possible participation of Sia during DENV cell internalization, and we observed the effect on DENV internalization, which was reduced in sialidase-pretreated samples (Figure 6(a), lanes 4 and 8). The internalization of DENV in CHO cells in the presence of different amounts of SG protein extract was dose dependent, as shown in Figure 5(b). These results support a general hypothesis that molecules in mosquito saliva and secretory SG proteins can potentiate pathogen-host transmission and that Sia residues play a role during DENV internalization in mammalian cells.


Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions.

Cime-Castillo J, Delannoy P, Mendoza-Hernández G, Monroy-Martínez V, Harduin-Lepers A, Lanz-Mendoza H, Hernández-Hernández Fde L, Zenteno E, Cabello-Gutiérrez C, Ruiz-Ordaz BH - Biomed Res Int (2015)

DENV-mammalian cells internalization assay. (a) DENV internalization by LLC-MK2 and CHO cells. The plot shows the internalization of [35S]-methionine-radiolabeled DENV by LLC-MK2 and CHO cells in the absence (lanes 2 and 6) and presence (lanes 3 and 7) of Ae. aegypti SG protein extract (SGH) and in the presence of SGH pretreated with sialidase before DENV incubation (lanes 4 and 8). DENV was mixed with SGH (from 80 SGs), which was pretreated or untreated with sialidase, before infecting mammalian cells with the DENV-SGH mixture. In the plot, the y-axis shows the counts per min of internalized DENV, *P < 0.05. (b) DENV internalization by CHO cells in the presence of different amounts of SGH. The plot shows that DENV internalization was enhanced by the presence of the protein extract from five SGs, which was dose dependent.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385653&req=5

fig6: DENV-mammalian cells internalization assay. (a) DENV internalization by LLC-MK2 and CHO cells. The plot shows the internalization of [35S]-methionine-radiolabeled DENV by LLC-MK2 and CHO cells in the absence (lanes 2 and 6) and presence (lanes 3 and 7) of Ae. aegypti SG protein extract (SGH) and in the presence of SGH pretreated with sialidase before DENV incubation (lanes 4 and 8). DENV was mixed with SGH (from 80 SGs), which was pretreated or untreated with sialidase, before infecting mammalian cells with the DENV-SGH mixture. In the plot, the y-axis shows the counts per min of internalized DENV, *P < 0.05. (b) DENV internalization by CHO cells in the presence of different amounts of SGH. The plot shows that DENV internalization was enhanced by the presence of the protein extract from five SGs, which was dose dependent.
Mentions: It is known that Ae. aegypti saliva enhances West Nile and Cache Valley virus infections, but it is unknown whether Aedes saliva can modulate DENV infections [6]. Based on our detection of interactions between DENV and salivary glycoproteins, we evaluated the possible participation of the Ae. aegypti SG protein extract in the modulation of DENV infection in different mammalian cell lines (LLCMK2 and CHO WT) using a DENV internalization assay, in the presence or absence of SG extracts. We found that DENV infection was enhanced in the presence of SG extract in both mammalian cell lines (Figure 6(a)). CHO cells appeared to be more permissive (fourfold enhancement; Figure 6(a), lane 7) than LLCMK2 (twofold enhancement; Figure 6(a), lane 3). We pretreated the SG protein extract with sialidase before the internalization assay to evaluate the possible participation of Sia during DENV cell internalization, and we observed the effect on DENV internalization, which was reduced in sialidase-pretreated samples (Figure 6(a), lanes 4 and 8). The internalization of DENV in CHO cells in the presence of different amounts of SG protein extract was dose dependent, as shown in Figure 5(b). These results support a general hypothesis that molecules in mosquito saliva and secretory SG proteins can potentiate pathogen-host transmission and that Sia residues play a role during DENV internalization in mammalian cells.

Bottom Line: AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface.Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome.Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology and Biotechnology Department, Biomedical Research Institute, National University of México (UNAM), 04510 México City, Mexico.

ABSTRACT
Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

Show MeSH
Related in: MedlinePlus