Endogenous ZAP is associated with altered Zika virus infection phenotype

Abstract

The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.

Keywords: Flavivirus; Interferon; RNA-seq; ZAP; Zika virus; Zinc finger antiviral protein.

Fig. 1

Infection phenotypes of Zika virus in wild-type (Vero-ZAP-WT) and ZAP knockout (Vero-ZAP-KO) cells. (A) ZAP Western blot. KO status was also confirmed by Sanger sequencing. Rabbit anti-ZAP IgG Abs (1:1,000 dilution; ANTI-ZC3HAV1; #HPA059096-100UL, Sigma-Aldrich), with the specific ZAP band appearing at 110 kDa, were used. Beta-actin was used as a western blot loading control (42 kDa). A normalized 50 µg of protein was used for all samples. Three replicates. Multiple bands may represent isoforms described for human ZAP [3] and still not experimentally characterized for monkeys. The full blot image is in Figure S2. (B) Zika virus NS5 protein Western blot. Reduced Zika virus NS5 protein loads in Vero-ZAP-WT cells as compared to Vero-ZAP-KO cells at 24 h and 72 h after inoculation, MOI 1. Rabbit anti-Zika virus NS5 IgG Abs (1:1,000 dilution; GTX133312, GeneTex), with the specific NS5 band appearing at 103 kDa, were used. Beta-actin was used as a western blot loading control (42 kDa). A normalized 50 µg of protein was used for all samples. Two biological replicates for each condition are shown. The full blot image is in Figure S3. (C) Zika virus infectious titers in supernatants of Vero-ZAP-WT and Vero-ZAP-KO cells at different times after inoculation, MOI 1. The experiment was done in four replicates for each cell line and sampling time. Supernatants from all mock-infected samples were negative. Statistics—an unpaired t-test. *A P-value of < 0.05 was considered statistically significant

Fig. 2

Significantly affected interferon signaling genes in different experimental conditions. (A) Mock-infected Vero-ZAP-WT versus Vero-ZAP-KO cells. Direction of analysis: WT/KO. Tables S3B,C. (B) Zika-infected Vero-ZAP-WT versus Vero-ZAP-KO cells. Direction of analysis: WT/KO. Tables S3D,E. (C) Mock-infected versus Zika-infected Vero-ZAP-WT cells. Direction of analysis: Zika-infected/Mock-infected. Tables S3F,G. (D) Mock-infected versus Zika-infected Vero-ZAP-KO cells. Direction of analysis: Zika-infected/Mock-infected. Tables S3H, I. Plots of the upregulated (red) and downregulated (blue) genes. FDR: The false discovery rate. Genes with FDR < 0.05 and log₂ fold change (FC) > 1 were significantly affected

Fig. 3

Biological “immune*” processes significantly affected in Vero-ZAP-WT and Vero-ZAP-KO cells at 24 h and 72 h after Zika virus infection. A Zika-infected Vero-ZAP-WT versus Vero-ZAP-KO cells. Direction of analysis: WT/KO. Tables S2G, H. B Mock-infected versus Zika-infected Vero-ZAP-WT cells. Direction of analysis: Zika-infected/Mock-infected. Tables S2K, L. C Mock-infected versus Zika-infected Vero-ZAP-KO cells. Direction of analysis: Zika-infected/Mock-infected. Tables S2O, P. Red bars–upregulated processes. Blue bars– downregulated processes. FDR: The false discovery rate