Identification of Host Factors for Rift Valley Fever Phlebovirus

Abstract

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID₅₀ assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Keywords: A549 cells; LACV; MP-12; RVFV; WDR7 gene; bunyavirus; host factor; phlebovirus.

Figure 1

Schematics of GeCKO-A549 cells generation, selection, NGS, and data analysis. A549 cells were transduced with the lentivirus-CRISPR-Cas9 library to generate GeCKO-A549 cells. Then, the GeCKO-A549 cells were subjected to three rounds of infection with the RVFV MP-12 (1 MOI) virus. The genomic DNA of round 0 GeCKO-A549 cells, round 1, and round 3 GeCKO-A549cells were sequenced using the Illumina NextSeq 550 platform. The output NGS data were analyzed using the MaGeCK program to generate the list of genes involved in RVFV replication.

Figure 2

Validation of gene hits via siRNA gene knockdown study. A549 cells were transfected with 50 nM of siRNAs. At 48 h post-transfection, the cells were infected with RVFV MP-12 virus at 0.1 MOI. At 24 h post-infection, the supernatant was collected and titered using plaque assay. NTC-non-target control siRNA; si46N-anti-RVFV siRNA; and WDR7, SLC35B2, EXOC4, LRP1, EMC3, CT47A1 gene-specific siRNAs were transfected. Each bar represents the average virus titer (pfu/mL) along with the corresponding standard deviation. Statistical analysis was performed on two independent experiments with four replicates for each, using the Mann–Whitney U test and independent Student’s t-test (** p-value < 0.005, *** p-value < 0.001).

Figure 3

Effect of WDR7 gene knockout (KO) on virus production of bunyaviruses: (A) A549 cells CT (non-knockout control) cells and WDR7 gene KO cell lines 1 and 2 were analyzed for WDR7 protein expression via Western blot using a WDR7-specific polyclonal antibody. (B–E) CT cells and WDR7 KO A549 cells were infected with RVFV MP-12 vaccine strain (B), with the wild-type RVFV Kenya 128B-15 strain (C), or with La Crosse encephalitis virus (D,E) at 0.1 MOI. The supernatant was collected at 6, 12 or 24 h post-infection (h pi) and titered using plaque assay (RVFV) or TCID₅₀-CPE assay (LACV). RVFV MP-12 testing on A549 CT cells and WDR7 KO lines 1 or 2, involved three to five independent experiments with three to four technical replicates each. RVFV Kenya 128B-15 testing involved independent experiments with three technical replicates each. LACV testing was performed in two independent experiments with eight technical replicates each. Statistical analysis was performed using the Mann–Whitney U test and independent Student’s t-test (* p-value < 0.05, ** p-value < 0.005, *** p-value < 0.001).

Figure 4

Viral RNA accumulation at various time points post-infection in WDR7 knockout (KO) cells. CT and WDR7 KO 1 cells were infected with (A) RVFV MP-12 vaccine strain or (B) LACV, both at 0.1 MOI. Total cellular RNA was harvested at various hours post-infection (h pi). One-step RT-qPCR was performed to detect the level of viral RNA using the PGK1 gene as an internal control. CT and WDR7 KO 1 cells were utilized. Each bar graph represents the average fold change in viral RNA expression, along with the corresponding standard deviation. Statistical analysis was performed on three independent experiments with two to three technical replicates for each, using the Mann–Whitney U test and independent Student’s t-test (* p-value < 0.05, *** p-value < 0.001, ns, non-significant).