Journal: Frontiers in Microbiology

Article Title: Deficient of glycosylation site in the envelop protein attenuated Zika virus replication in mosquito cells

doi: 10.3389/fmicb.2025.1603083

Figure Lengend Snippet: T156I mutation delays the replication of ZIKV in mosquito cells. (A,B) The wild type (WT) and T156I mutant (T156I) virus were subjected to infect mosquito C6/36 cells. The whole cell lysates were collected after 12–72 hpi to determine the expression level of E protein using western blotting assay. The GAPDH were used as the house-keeping gene for normalization. The grayscale of the bands in western blotting assay were analyzed by ImageJ software. Asterisks indicate significant differences at 24 hpi between cells infected by WT (orange column) and cells infected by T156I (green column). (C) Mosquito cells were infected by WT or T156I virus for 12 hpi and 72 hpi. E protein (green) and cell nucleic (DAPI, blue) were immuno-stained to analyze the expression of E protein. (D,E) Chloroquine and MG132 were added to the cells followed by WT or T156I infection. The whole cell lysates were collected after 24 hpi to determine the expression level of E protein using western blotting assay. The GAPDH were used for normalization. (F,G) Mosquito cells were infected by WT or T156I. The cell lysates were used to extract the total RNA and subjected to detect the mRNA level of R protein. The supernatants were used to determine the virus titer by plaque-forming assay in VeroE6 cells. (H) The morphology of plaques was scanned. All the results represent the mean value ± standard error of the mean pooled from three independent experiments with duplicated samples. Asterisks indicate significant differences between cells infected by WT (orange column) and cells infected by T156I (green column). Statistical analysis was performed with unpaired Student’s t -test, * p < 0.01, ** p < 0.005, *** p < 0.001.

Article Snippet: The membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibody against Zika virus (ZIKV) envelope protein (SinoBiological, 40543-R029) overnight at 4 °C.

Techniques: Mutagenesis, Virus, Expressing, Western Blot, Software, Infection, Staining

Journal: Frontiers in Microbiology

Article Title: Deficient of glycosylation site in the envelop protein attenuated Zika virus replication in mosquito cells

doi: 10.3389/fmicb.2025.1603083

Figure Lengend Snippet: T156I mutation potentially weaken the stability of E-dimer but not antibody receptor binding affinity (A) The molecular simulation of the T156I mutation on the structure of ZIKV E protein. The red, yellow, and blue colors represent the domain I, domain II and domain III of the E protein which mediate the formation of E-dimer. (B) The protein model was evaluated using Ramachandran plot analysis. The results indicate that 92.3% of the amino acids are located in the most favorable region, 7.1% in additional allowed regions, 0.3% in generously allowed regions, and 0.3% in disallowed regions-totaling 100%, which confirming the reliability of the E protein structural model. (C) The potential interaction patterns at the 156I residue with neighboring amino acids. Yellow stick represents the hydrogen bonds, pink stick showed the weak C–H bonds, and the gray stick represent the two sets of hydrophobic interactions with surrounding residues. (D) The HDCOK program was used to perform a global docking simulation of the binding interface and interaction mode between the 156I mutant E protein and the ZIKV neutralizing antibody EDE2. (E) Revealed that the mutant complex forms seven sets of hydrogen bonds (gray), four sets of weak C–H bonds (yellow), and two sets of hydrophobic interactions (pink). (F) The potential interactions of I156 with surrounding residues in 2D analysis.

Article Snippet: The membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibody against Zika virus (ZIKV) envelope protein (SinoBiological, 40543-R029) overnight at 4 °C.

Techniques: Mutagenesis, Binding Assay, Residue

Journal: bioRxiv

Article Title: Heparanase attenuates Zika virus infection by destabilizing the viral envelope protein

doi: 10.1101/2025.03.16.643511

Figure Lengend Snippet: ( A ) FLAG-E was co-transfected with Hpa, Hpa-DM, or vector to HEK-293 cells separately. The expression of ZIKV E protein and Hpa or Hpa-DM were examined by western blot. One representative experiment of three was shown. ( B ) The intensity of ZIKV E protein and GAPDH specific bands were quantified using Image J software. The relative intensity of the envelope was calculated as the ratio of FLAG-E and GAPDH densitometry values. The data were presented as fold change by normalizing to the relative intensity of FLAG-E in the vector control condition. Mean ± SD from three experiments was calculated. Statistical analysis was performed using Student’s t-test. **p≤ 0.01. ( C ) FLAG-NS1 was co-transfected with Hpa, Hpa-DM, or vector to HEK-293 cells separately. The expression of NS1 protein and Hpa or Hpa-DM was assessed by western blot.

Article Snippet: The following antibodies were used in this study: Monoclonal ANTI-FLAG® M2 antibody (1:5000, Sigma, F1804); Polyclonal anti-heparanase S63 (1:1000, 1:200IF) was kindly provided by Prof. Vlodavsky (Technion Integrated Cancer Center, Haifa, Israel); Ubiquitin Antibody (P4D1) (1:1000, Santa Cruz, sc-8017); GAPDH Monoclonal antibody (1:10000, Proteintech, 60004-1-Ig); Zika virus Envelope protein antibody (1:2000, Gene Tex, GTX133314); Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, 31430); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, G21234), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Invitrogen, A-11012).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Software, Control

Journal: bioRxiv

Article Title: Heparanase attenuates Zika virus infection by destabilizing the viral envelope protein

doi: 10.1101/2025.03.16.643511

Figure Lengend Snippet: FLAG-E was co-transfected with Hpa or vector. After 40 h, the cells were treated with or without MG132 ( A ) or NH 4 Cl ( C ) for 8h. The expression of FLAG-E and Hpa were evaluated by western blot using anti-FLAG or anti-Hpa antibodies separately. One experiment of three was shown here. ( B ) or ( D ) The intensity of ZIKV E protein and GAPDH specific bands in (A) or (C) were quantified using Image J. The relative expression of FLAG-E was calculated by the intensity ratio of FLAG-E / GAPDH and normalized to the value in the co-transfection of FLAG-E with vector without treatment. The data was shown as mean ± SD from three experiments. Statistical analysis was performed using Student’s t-test. **p≤ 0.01. ( E ) HA-Ub was co-transfected with FLAG-E and untagged Hpa. Cells were treated with MG132 (10µM) for 6h before the immunoprecipitation with anti-FLAG beads in the denature conditions and the blots were probed with an anti-Ub antibody. The intensity of ZIKV E Ub conjugates was normalized by the intensity of the ZIKV E specific bands separately.

Article Snippet: The following antibodies were used in this study: Monoclonal ANTI-FLAG® M2 antibody (1:5000, Sigma, F1804); Polyclonal anti-heparanase S63 (1:1000, 1:200IF) was kindly provided by Prof. Vlodavsky (Technion Integrated Cancer Center, Haifa, Israel); Ubiquitin Antibody (P4D1) (1:1000, Santa Cruz, sc-8017); GAPDH Monoclonal antibody (1:10000, Proteintech, 60004-1-Ig); Zika virus Envelope protein antibody (1:2000, Gene Tex, GTX133314); Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, 31430); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, G21234), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Invitrogen, A-11012).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Cotransfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Heparanase attenuates Zika virus infection by destabilizing the viral envelope protein

doi: 10.1101/2025.03.16.643511

Figure Lengend Snippet: ( A ) Huh7 or ( B ) HEK-293 stably overexpressing Hpa or Hpa-DM cell lines were infected by ZIKV (MOI=0.1). After 48h (for Huh7) and 24 h (for HEK-293), the expression of E protein was assessed by western blot using the specific antibody. ( C ) MEF or MEF-Hpa-KO cells were infected with ZIKV (MOI=0.1). The expression of ZIKV E protein was assessed by western blot at 48h post-infection. ( D ) The replication of ZIKV was evaluated by qPCR using the specific primers targeting the ZIKV E gene. The results were presented as fold change relative to the infection of Huh7-Vec cells and the data were shown as mean ± SD from two experiments. ( E ) or ( F ) The titer of virus particles released into the supernatant in ( A ) or ( C ) was measured by standard plaque assay. The number of plaques was normalized by vector control or wild-type cells and was expressed as mean ± SD of two or three experiments. Statistical analysis was performed using Student’s t-test. *p≤ 0.05, **p≤ 0.01.

Article Snippet: The following antibodies were used in this study: Monoclonal ANTI-FLAG® M2 antibody (1:5000, Sigma, F1804); Polyclonal anti-heparanase S63 (1:1000, 1:200IF) was kindly provided by Prof. Vlodavsky (Technion Integrated Cancer Center, Haifa, Israel); Ubiquitin Antibody (P4D1) (1:1000, Santa Cruz, sc-8017); GAPDH Monoclonal antibody (1:10000, Proteintech, 60004-1-Ig); Zika virus Envelope protein antibody (1:2000, Gene Tex, GTX133314); Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, 31430); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, G21234), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Invitrogen, A-11012).

Techniques: Stable Transfection, Infection, Expressing, Western Blot, Virus, Plaque Assay, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Heparanase attenuates Zika virus infection by destabilizing the viral envelope protein

doi: 10.1101/2025.03.16.643511

Figure Lengend Snippet: ( A ) Huh7 cells were infected by ZIKV (MOI=0.1). Cells were harvested at different time points after infection as shown in the figure. The expression of ZIKV E protein was examined by western blot to monitor ZIKV infection. ( B ) The expression of HPSE at the mRNA level was quantified by qPCR. The change of HPSE mRNA was calculated as fold change related to the mock infection and results were shown as mean ± SD from three experiments. Statistical analyses were performed by one-way ANOVA. *p≤ 0.05. ( C ) The expression of GRP78 at the mRNA level was evaluated by qPCR at 48h post-ZIKV infection from the samples in (B).

Article Snippet: The following antibodies were used in this study: Monoclonal ANTI-FLAG® M2 antibody (1:5000, Sigma, F1804); Polyclonal anti-heparanase S63 (1:1000, 1:200IF) was kindly provided by Prof. Vlodavsky (Technion Integrated Cancer Center, Haifa, Israel); Ubiquitin Antibody (P4D1) (1:1000, Santa Cruz, sc-8017); GAPDH Monoclonal antibody (1:10000, Proteintech, 60004-1-Ig); Zika virus Envelope protein antibody (1:2000, Gene Tex, GTX133314); Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, 31430); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, G21234), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Invitrogen, A-11012).

Techniques: Infection, Expressing, Western Blot