A Human and Rhesus Macaque Interferon-Stimulated Gene Screen Shows That Over-Expression of ARHGEF3/XPLN Inhibits Replication of Hepatitis C Virus and Other Flavivirids

Abstract

Natural hepatitis C virus (HCV) infection is restricted to humans, whereas other primates such as rhesus macaques are non-permissive for infection. To identify human and rhesus macaque genes that differ or share the ability to inhibit HCV replication, we conducted a medium-throughput screen of lentivirus-expressed host genes that disrupt replication of HCV subgenomic replicon RNA expressing secreted Gaussia luciferase. A combined total of >800 interferon-stimulated genes (ISGs) were screened. Our findings confirmed established anti-HCV ISGs, such as IRF1, PKR and DDX60. Novel species−specific inhibitors were also identified and independently validated. Using a cell-based system that recapitulates productive HCV infection, we identified that over-expression of the ‘Rho Guanine Nucleotide Exchange Factor 3’ gene (ARHGEF3) from both species inhibits full-length virus replication. Additionally, replication of two mosquito-borne flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV), were also reduced in cell lines over-expressing ARHGEF3 compared to controls. In conclusion, we ascribe novel antiviral activity to the cellular gene ARHGEF3 that inhibits replication of HCV and other important human viral pathogens belonging to the Flaviviridae, and which is conserved between humans and rhesus macaques.

Keywords: ARHGEF3; Flaviviridae; HCV; Hepatitis C Virus; ISG; XPLN; interferon; interferon stimulated gene.

Figure 1

Screening for ISGs from humans and rhesus macaques that influence HCV-SGR luciferase reporter activity. (A) Schematic depiction of the screening protocol used in the study showing that Huh7 cells were transduced and transfected with luciferase-expressing HCV SGR after 48 h and the secreted luciferase levels were quantified at 72 h post-transfection (hpt). (B) Dot plot of RLU detected for ISG-expressing cells transfected with HCV-SGR RNA for human (Hs; green) and rhesus macaque (Mm; red) ISGs, relative to the control ISGs (%) from 72 hpt. The primary screen was performed as single assays. Genes of interest are marked on the figure. Limit of detection (L.O.D) highlighted with a dotted line.

Figure 2

Secondary screen validation of anti-HCV-SGR ISGs from humans and rhesus macaques detected in the initial screen. (A,B) RLU detected at 72 hpt in HCV-SGR RNA (wild-type [WT]) transfected cells expressing human (A) or rhesus macaque (B) ISGs. (C,D) Validation of anti-HCV-SGR activity of human and rhesus macaque ISGs against WT (C), or replication-defective HCV-SGR RNA (D) at 72 hpt. The ratio of WT:GND RLU (E) and HCV RNA levels (F) was calculated at 72 hpt. For the data in panel F, WT HCV-SGR RNA was measured by RT-qPCR following electroporation of SGR RNA into stable puromycin-selected ISG-expressing Huh7 cell lines. Negative controls are shown (black) and ISGs that gave a >2-fold reduction in HCV RNA replication are indicated (pink). ISGs that do not achieve this threshold are shown in green. Values from triplicate wells as technical replicates were used and variation is shown as standard error of the mean.

Figure 3

Validation of ISGs with activity against HCVcc. The effect of ISG expression on replication of full-length Jc1-HCVcc was ascertained following infection of stably transduced ISG-expressing cell lines by quantification of HCV viral RNA abundance by RT-qPCR at 72 hpi (moi = 0.1). Negative controls are shown (black) and ISGs that reached a threshold of >2-fold reduction in viral replication are shown in pink. ISGs that do not achieve this threshold are shown in green. Values from combined duplicate technical replicates from three independent experiments were used and variation is shown as standard error of the mean.

Figure 4

Validation of the antiviral activity of ARHGEF3/XPLN on HCV RNA replication. (A) Relative HCV RNA abundance from HCVcc infection of Huh7 cells expressing ARHGEF3 from Human (Hs) or rhesus macaque (Mm) as compared to negative control at 72 hpi. (B) Relative RLU (%) following HCV SGR RNA transfection of Huh7 cells expressing ARHGEF3 from Human (Hs) or rhesus macaque (Mm) as compared to negative control at 72 hpt. (C) Effect of ARHGEF3/XPLN-specific mutations (Myc-tagged WT, N-terminus truncation, N-terminus only and the GEF-inactivation mutant W440L) on HCV RNA replication following SGR RNA transfection. (D) Schematic of ARHGEF3 showing the N (amino acids 1–125), diffuse B cell lymphoma homology (DH) and plekstrin homology (PH) domains, alongside the location of the W440 residue. RLU activity was measured and compared to controls at 72 hpt. Values from combined three technical replicates from two independent experiments were used and variation is shown as standard error of the mean.

Figure 5

Antiviral activity of ARHGEF3 against flaviviruses. RLU activity in Huh7 cells stably expressing ARHGEF3/XPLN or IRF1 following infection with recombinant luciferase-expressing YFV (A) and ZIKV (B) as measured at 48 hpi and 72 hpi, respectively. Values from 3 technical replicates for YFV and 9 technical replicates for ZIKV were used and variation is shown as standard deviation of the mean alongside p values for Student’s T test between EMPTY and ARHGEF3 WT results.

Figure 6

Abundance of ARHGEF3/XPLN RNA in vivo in HCV-infected human liver biopsies. RNA levels (FKPM) of known anti-HCV ISGs in liver biopsies from uninfected individuals (n = 4) (A). Fold-change in RNA (FPKM) levels of known ISGs in liver biopsies from infected individuals (HCV gt1: pink; gt3: green (each n = 5)) compared to uninfected controls (B).