Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection

Abstract

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

Keywords: Zika virus; bortezomib; quantitative proteomics; ubiquitin proteasome system; virus-host interaction.

FIG 1

Quantitative proteomic analysis of ZIKV-infected C6/36 cells. (A) Kinetics of ZIKV replication in C6/36 cells. C6/36 cells were infected with ZIKV strain SZ-WIV01 at an MOI of 1 and were harvested at the indicated time points. Intracellular RNA was extracted and subjected to reverse transcription. The relative intracellular level of ZIKV RNA was measured by quantitative PCR. All ratios are relative to ZIKV RNA level at 3 hpi. All experiments were performed at least three times, and the values represent the means ± SDs from three replicates. The RNA level at 120 hpi was still quantifiable, although CPE can be detected at this time point. (B) Workflow for quantitative proteomic analysis of ZIKV-infected C6/36 cells. (C) The correlation of protein ratios from three biological replicates. Pearson correlation efficiency (Corr.) was calculated based on the log₂ values (protein ratio) from two independent biological replicates. (D) The Gaussian distribution of protein ratios, quantified. Gaussian distribution of protein ratios was analyzed, and proteins with ratios deviating from the mean of the normally distributed data by 1.96 SDs were considered differentially regulated.

FIG 2

Validation of MS results using quantitative PCR. C6/36 cells were infected with ZIKV SZ-WIV01 at an MOI of 1 or mock treated. At 96 hpi, cells were harvested and intracellular mRNAs were extracted and subjected to reverse transcription. The intracellular RNA levels of proteins were measured by quantitative PCR. The putative glyceraldehyde-3-phosphate dehydrogenase was chosen as the internal control. All quantitative PCRs were performed at least three times, and the values represent the means and SDs from three replicates. Protein ratio quantified by MS is presented alongside the protein ratio quantified by quantitative PCR. (A) Quantitative PCR analysis indicated that these proteins are upregulated by ZIKV infection in C6/36 cells (P < 0.05). (B) Quantitative PCR analysis indicated that these proteins are downregulated by ZIKV infection in C6/36 cells (P < 0.05).

FIG 3

GO analysis of quantified proteins in ZIKV-infected C6/36 cells using a statistical enrichment test. Quantified proteins with their gene symbols and ratios were submitted to PANTHER to perform a statistical enrichment test, which can assess whether the numeric values of protein ratios in certain biological process are nonrandomly distributed with respect to the numeric values of all quantified protein ratios (overall). Biological process categories with P values of <0.05 were considered regulated significantly.

FIG 4

CHCHD2 promotes ZIKV replication. (A) ZIKV infection upregulates the intracellular mRNA level of a CHCHD2-like protein in C6/36 cells. C6/36 cells preseeded were infected with ZIKV at an MOI of 1 or mock treated. At the indicated time points, ZIKV-infected and mock-treated cells were harvested. Intracellular mRNA was extracted and subjected to reverse transcription. The relative mRNA level of the protein with accession number KXJ75878 (CHCHD2-like protein) was measured by quantitative PCR, a GAPDH-like protein was set as the endogenous control, and a mock-treated control sample was set as the reference (mRNA level set as 1). (B) ZIKV infection upregulates the intracellular mRNA level of CHCHD2 in HeLa cells. HeLa cells were infected with ZIKV at an MOI of 1. The intracellular mRNA level of CHCHD2 was measured with the method described above. (C) Western blot analysis of CHCHD2 expression in HeLa cells. HeLa cells were transfected with siRNA against CHCHD2 or plasmid expressing HA-CHCHD2. At 36 h posttransfection, cells were harvested and subjected to WB analysis. Antibodies used are indicated, with GAPDH set as the endogenous loading control. (D) Overexpression of HA-CHCHD2 promotes ZIKV production in TCS. HeLa cells were transfected with plasmid expressing HA-CHCHD2, and 36 h posttransfection, cells were infected with ZIKV strain H/PF/2013 at an MOI of 1. At 12 hpi and 36 hpi, TCS was collected and virus titer was measured with plaque assay. (E) Knockdown of CHCHD2 decreases ZIKV production in TCS. HeLa cells were transfected with siRNA against CHCHD2, and 36 h posttransfection, cells were infected with ZIKV strain H/PF/2013 at an MOI of 1. At 12 hpi and 36 hpi, TCS was collected and virus titer was measured with a plaque assay. (F) Western blot analysis of CHCHD2 expression in HEK 293T cells. HEK 293T cells were transfected with siRNA against CHCHD2 or plasmid expressing HA-CHCHD2. At 24 h posttransfection, cells were harvested and subjected to WB analysis. Antibodies used are indicated, with GAPDH set as the endogenous loading control. (G) Overexpression of HA-CHCHD2 inhibits SeV-induced IFN-β promoter activity. HEK 293T cells were transfected with reporter plasmids and the indicated plasmids and were infected with SeV at 36 h posttransfection. At 12 hpi, cells were lysed to measure intracellular luciferase activity. (H) Knockdown of CHCHD2 promotes SeV-induced IFN-β promoter activity. HEK 293T cells were transfected with reporter plasmids and the indicated siRNAs and were infected with SeV at 36 h posttransfection. At 12 hpi, cells were lysed to measure intracellular luciferase activity. All experiments were performed at least three times, and the values represent the means and SDs from three replicates. *, P < 0.05; **, P < 0.01.

FIG 5

Bortezomib affects ZIKV infection in a dose-dependent manner. (A) Evaluation of CC₅₀ levels for bortezomib in C3/36 cells. Preseeded C3/36 cells were treated with increasing concentrations of bortezomib for 24 h and then subjected to cell viability analysis. (B) Effects of bortezomib on ZIKV SC-WIV01 replication in C6/36 cells. C3/36 cells were treated with bortezomib and infected with ZIKV strain SC-WIV01 at an MOI of 1; at 24 hpi, the intracellular level of ZIKV RNA was measured by quantitative PCR. (C) Effects of bortezomib on ZIKV strain H/PF/2013 replication in C6/36 cells. C3/36 cells were treated with bortezomib and infected with ZIKV strain H/PF/2013 at an MOI of 1; at 24 hpi, the intracellular level of ZIKV RNA was measured by quantitative PCR. (D) Bortezomib affects ZIKV H/PF/2013 replication in C6/36 cells in a dose-dependent manner. C6/36 cells were treated with the indicated concentrations of bortezomib and infected with ZIKV strain H/PF/2013 at an MOI of 1. At 24 hpi, C6/36 cells were fixed and incubated with mouse anti-flavivirus group antigen antibody followed by a DyLight488-labeled antibody to mouse IgG to detect ZIKV E protein (green). Then the intracellular level of ZIKV E protein in C6/36 cells was detected by immunofluorescence analysis (IFA). (E) Evaluation of CC₅₀ levels for bortezomib in Vero cells. Preseeded Vero cells were treated with increasing concentrations of bortezomib for 24 h and then subjected to cell viability analysis. (F) Bortezomib affects ZIKV H/PF/2013 replication in Vero cells in a dose-dependent manner. Vero cells were treated with the indicated concentrations of bortezomib and infected with ZIKV strain H/PF/2013 at an MOI of 1. At 24 hpi, TCS was collected and viral titer was measured with a plaque assay. (G) Bortezomib affects ZIKV strain H/PF/2013 replication in HeLa cells in a dose-dependent manner. (H) Bortezomib affects ZIKV H/PF/2013 strain replication in HFFs in a dose-dependent manner. (I) Bortezomib affects ZIKV infection at the early stage. Vero cells were treated with 25.6 nM bortezomib at different time points prior to or after the addition of ZIKV strain H/PF/2013 at an MOI of 1. After incubation with bortezomib and virus for 2 h, cells were washed and cultured in DMEM with 10% FBS. At 24 hpi, cells were harvested and the intracellular level of ZIKV RNA was quantified by quantitative PCR. hbi, hours before infection. All experiments were performed at least three times, and the values represent the means and SDs from three replicates. *, P < 0.05; **, P < 0.01.

FIG 6

MG132 affects the ZIKV entry process. (A) Evaluation of CC₅₀ levels for MG132 in Vero cells. Preseeded Vero cells were treated with increasing concentrations of MG132 for 24 h and then subjected to cell viability analysis. (B) MG132 affects ZIKV strain H/PF/2013 replication in Vero cells in a dose-dependent manner. Vero cells were treated with the indicated concentrations of MG132 and infected with ZIKV strain H/PF/2013 at an MOI of 1. At 24 hpi, TCS was collected and viral titers were measured with a plaque assay. (C) MG132 affects ZIKV strain H/PF/2013 replication in HeLa cells in a dose-dependent manner. (D) MG132 affects ZIKV strain H/PF/2013 replication in HFFs in a dose-dependent manner. (E) MG132 affects ZIKV infection at the early stage. Vero cells were treated with 12.8 μM MG132 at different time points prior to or after the addition of ZIKV strain H/PF/2013 at an MOI of 1. After incubation with MG132 and virus for 2 h, cells were washed and cultured in DMEM with 10% FBS. At 24 hpi, cells were harvested and the intracellular level of ZIKV RNA was quantified with quantitative PCR. All experiments were performed at least three times, and the values represent the means and SDs from three replicates. *, P < 0.05; **, P < 0.01.

FIG 7

Bortezomib reduced the viral load and signs of ZIKV pathology in mice. WT mice treated with Ifnar-blocking mouse MAb were infected intraperitoneally with ZIKV and were treated with bortezomib (1 mg/kg) in PBS or with PBS (vehicle control). Mice were analyzed at days 7 and 14 after infection. (A) Body weights of mice. Results are pooled from 6 mice in each group. (B) Viral loads in the brain and testis. Testis and brain were collected at days 7 and 14 after ZIKV infection, and viral load was determined with a plaque assay. At day 7, viral loads in brain and testis were lower in bortezomib-treated mice, and at day 14, viral loads in testis were lower in bortezomib-treated mice (n = 6; Student's t test; **, P < 0.01). (C) Histology of the testis at day 14 after ZIKV infection. At day 14 after ZIKV infection, a slight loss of germ cells can be observed in testis from vehicle-treated mice (indicated by arrow). Representative images from several independent experiments are shown.

FIG 8

Global view of host proteins regulated in ZIKV-infected C6/36 cells. GO descriptions of all regulated proteins were referred to their annotations in NCBI, and gene symbols of regulated proteins were assigned based on the best match derived from the alignments with NCBI database protein reference sequences. Regulated host proteins are sorted and aligned according to their biological functions. A large number of regulated proteins are involved in vesicular transport, mitochodria, and replication, transcription, and translation and are annotated in Table S3.