APOBEC3G is a restriction factor of EV71 and mediator of IMB-Z antiviral activity

Highlights

• A3G restricts EV71 replication in a deaminase activity-independent manner.

• IMB-Z inhibits EV71 replication by elevating the level of cellular A3G.

• IMB-Z promotes the endogenous A3G packaging into EV71 virion and reduces its infectivity.

Abstract

Enterovirus 71 (EV71), a single-stranded positive-sense RNA virus, is the causative agent of hand, foot, and mouth disease (HFMD), for which no effective antiviral therapy is currently available. Apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a cytidine deaminase that inhibits the replication of several viruses, such as human immunodeficiency virus-1, hepatitis B virus and hepatitis C virus. In our efforts toward understanding the antiviral spectrum and mechanism of A3G, we found that ectopic expression of A3G inhibited EV71 replication, whereas knockdown of endogenous A3G expression promoted EV71 replication. Moreover, inhibition of EV71 replication by IMB-Z, a N-phenylbenzamide derivative, is associated with increased levels of intracellular A3G, but reducing the level of A3G by RNA interference diminished the antiviral activity of IMB-Z. Mechanistically, we obtained evidence suggesting that the cytidine deaminase activity is not required for A3G inhibition of EV71 replication. Instead, we demonstrated that A3G can interact with viral 3D RNA-dependent RNA polymerase (RdRp) and viral RNA and be packaged into progeny virions to reduce its infectivity. Taken together, our results indicate that A3G is a cellular restriction factor of EV71 and mediator of the antiviral activity of IMB-Z. Pharmacological induction and/or stabilization of A3G is a potential therapeutic approach to treat diseases caused by EV71 infection, such as HFMD.

Introduction

Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus belonging to the enterovirus genus of the Picornaviridae family. Although EV71 infection usually results in mild clinical symptoms and is self-limited, severe EV71 infection is often associated with neurological diseases, including aseptic meningitis, brain stem encephalitis, and acute flaccid paralysis (Wang et al., 2012a, 2017; Wu et al., 2013). EV71 infection also causes hand, foot, and mouth disease (HFMD) in children, mostly under 5 years of age. Since the first reported case of EV71 infection in California in 1969, EV71 outbreaks have been periodically reported worldwide, especially in the Asia-Pacific region (Cardosa et al., 2003; Chen et al., 2017; Huang et al., 2008; Wang et al., 2013). Hundreds of cases involving lethal complications have been reported in each outbreak. In China, EV71 caused a severe HFMD outbreak in 2008 and has now become a serious threat to children's health (Chen et al., 2017; He et al., 2017). Millions of children are infected with EV71 each year, and the morbidity and severity of HFMD have increased annually. However, no specific antiviral drug is currently available for treatment of EV71 infections.

APOBEC3G (apolipoprotein B messenger RNA [mRNA]-editing enzyme catalytic polypeptide-like 3G [A3G]), a member of the APOBEC superfamily, is an interferon-inducible cellular protein and plays an important role in defending viral infections. A3G is a cytidine deaminase containing a conserved His-X-Glu and Cys-X-X-Cys Zn²⁺ coordination motif and has been demonstrated to restrict the infection of several viruses, including human immunodeficiency virus-1 (HIV-1), T-cell leukemia virus type 1 (HTLV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) (Bishop et al., 2008; Chiu and Greene, 2008; Köck and Blum, 2008; Olson et al., 2018; Peng et al., 2011; Smith et al., 2012; Sasada et al., 2005; Zhu et al., 2015). Mechanistically, A3G inhibits retroviral replication by either cytidine deamination of viral DNA, which results in G-to-A hypermutation of viral genomes (Goila-Gaur and Strebel, 2008; Okada and Iwatani, 2016), or disruption of reverse transcription or genome encapsidation in a deaminase activity-independent manner (Fehrholz et al., 2012; Nguyen and Hu, 2008). However, A3G inhibition of HCV replication via interaction with viral NS3 protein in a deaminase activity independent manner (Zhu et al., 2015). Li et al. recently reported that A3G binds to the 5′UTR of EV71 to inhibit viral protein translation and genome replication. Intriguingly, EV71 antagonizes the restriction of A3G through its non-structural protein 2C that induces the autophagy–lysosome degradation of A3G (Li et al., 2018). Moreover, compound IMB-26 was reported to directly bind to and stabilize A3G (Cen et al., 2010). In our previous study, a series of N-phenylbenzamide derivatives of IMB-26 had been synthesized, and their anti-EV71 activities were assayed in vitro (Ji et al., 2013). Among the compounds tested, compound 5b (N-(4-chlorophenyl)-4-methoxy- 3-propionamidobenzamide, C₁₇H₁₇N₂O₃Cl, MW 332.78, Fig. 1A, renamed to IMB-Z) presented an improved antiviral activity against EV71 (Ji et al., 2013). In this study, we investigated the possibility that IMB-Z inhibition of EV71 replication is due to its elevation of cellular A3G.