Suramin inhibits chikungunya virus replication through multiple mechanisms
Introduction
Chikungunya virus (CHIKV) is a mosquito-borne arthritogenic alphavirus that has infected millions of people since its re-emergence in 2005. In November 2013, CHIKV emerged in the Caribbean (Weaver, 2014, Weaver and Lecuit, 2015), starting an outbreak that has thus far resulted in over 1.2 million cases in the Americas (http://www.paho.org/hq/index.php?Itemid=40931).
CHIKV replication occurs in the cytoplasm on modified endosomal membranes and is driven by replication and transcription complexes (RTCs) that contain CHIKV nonstructural proteins (nsP) nsP1-4, of which nsP4 is the RNA-dependent RNA polymerase (RdRp). Early in infection negative-stranded RNA (−RNA) complementary to the viral genome is synthesized, which serves as template for the production of genomic and subgenomic RNA (sgRNA). The genome serves as mRNA for the production of nsPs and the sgRNA is translated into the structural proteins that are required for the biogenesis of new virions.
Despite intensified research efforts over the past years and the identification of a variety of compounds with anti-CHIKV activity in preclinical studies (Thiberville et al., 2013), there are still no registered drugs on the market for treating CHIKV infections. Suramin is a symmetrical sulfonated naphthylurea compound that was approved for the treatment of parasitic infections in 1921, but its anti-cancer and antiviral potential were discovered only 60 years later (reviewed in De Clercq, 2015, Liu and Zhuang, 2011, Voogd et al., 1993). It was shown that suramin had anti-reverse transcriptase activity against tumor-inducing viruses (De Clercq, 1979) and it was actually the first documented HIV reverse transcriptase inhibitor that was tested in human patients (Broder et al., 1985), but the compound’s side effects outweighed the clinical benefit due to the required long term treatment (Kaplan et al., 1987). A later study revealed that suramin’s anti-HIV-1 activity in vivo was actually due to its inhibitory effect on the interaction between the viral gp120 and the CD4 receptor (Schols et al., 1990). Suramin has also been shown to block the binding or early steps of infection of several DNA and RNA viruses, like herpes simplex virus type-1 (Aguilar et al., 1999), cytomegalovirus (Baba et al., 1993), human hepatitis B virus (Schulze et al., 2007), hepatitis delta virus (Petcu et al., 1988), hepatitis C virus (Garson et al., 1999), dengue virus (Chen et al., 1997), several bunyaviruses (Crance et al., 1997, Ellenbecker et al., 2014, Iqbal et al., 2000, Jiao et al., 2013), norovirus-like particles (Tamura et al., 2004) and enterovirus 71 (Wang et al., 2014), for which the antiviral activity of suramin was also confirmed in an animal model (Ren et al., 2014). In recent in vitro studies suramin was identified as a hepatis C virus and dengue virus helicase inhibitor (Basavannacharya and Vasudevan, 2014, Mukherjee et al., 2012) and also as a norovirus RdRp inhibitor by virtual screening and biochemical assays with purified enzymes (Mastrangelo et al., 2012, Tarantino et al., 2014). In the present study we assessed the effect of suramin on CHIKV RNA synthesis using our recently established in vitro assay that relies on RTCs isolated from infected cells (Albulescu et al., 2014). We found that suramin inhibits both CHIKV RNA synthesis in vitro as well as an early step in CHIKV infection of cultured cells. In addition to describing the inhibition of CHIKV replication through two independent mechanisms, we provide more insight into the moieties required for suramin’s antiviral activity.
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Cell lines, viruses and virus titration
Vero E6 and BHK-21 cell culture and infectious clone-derived CHIKV LS3 and strain ITA07-RA1 have been described previously (Scholte et al., 2013). CHIKV STM35 is an infectious clone-derived virus based on the sequence of a clinical isolate from the island of St. Martin (manuscript in preparation). CHIKV M5 is a reverse-engineered LS3-derived (nsP4) mutant virus that is resistant to favipiravir (Delang et al., 2014) and CHIKV C483Y is identical to LS3 except for a C483Y mutation in nsP4 that
Suramin inhibits RNA synthesis of CHIKV and other alphaviruses in vitro
As suramin was previously shown to inhibit the in vitro activity of a number of viral polymerases, including that of noroviruses (Mastrangelo et al., 2012, Tarantino et al., 2014), we set out to study its effect on CHIKV RNA synthesis using our recently established in vitro assay that is based on the RNA-synthesizing activity of RTCs isolated from CHIKV-infected cells. This assay measures the incorporation of [α]32P-CTP into viral RNA, which was severely impaired by suramin in a dose-dependent
Conclusion
In this study we show that the anti-parasitic drug suramin inhibits the replication of CHIKV and other alphaviruses. We discovered that while in vitro suramin is a potent inhibitor of RNA synthesis, in cell culture the compound mainly inhibits an earlier, post-attachment step of the CHIKV replicative cycle, likely viral entry. The inhibition of an early step in infection (receptor binding or entry or uncoating) has also been reported for a variety of other viruses (Aguilar et al., 1999, Baba et
Acknowledgements
This work was supported by the EU-FP7 EUVIRNA (# 264286) and SILVER (# 260644) Grants, as well as by the Ministry of Science and Technology of R.O.C. (# NSC 103-2923-I-008-001 and MOST 103-2113-M-007-018-MY3) and the National Central University, Taiwan (# 103G603-14). We are grateful to the LUMC biosafety officer dr. Gijsbert van Willigen for his support in keeping our BSL-3 facility operational and to dr. Gilles Querat (Aix Marseille University) for supplying CHIKV RNA isolated during the
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