Polysulfonate suramin inhibits Zika virus infection

Highlights

• Polysulfonate suramin significantly inhibits ZIKV infection.

• Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication.

• Molecular dynamics simulation revealed strong interaction of suramin with ZIKV helicase.

• Zika virus does not bind to cell surface heparan sulfate.

Abstract

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3–5 log₁₀ PFU viral reduction with IC₅₀ value of ∼2.5–5 μg/ml (1.93 μM–3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1–24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.

Introduction

Zika virus (ZIKV) is an arthropod-borne RNA virus in the genus of flavivirus and the family of Flaviviridae. The Flaviviridae family also contains other vector-borne viruses relevant to human health, such as dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus. ZIKV was first isolated from a febrile sentinel rhesus macaque in 1947 and from an Aedes africanus mosquito in 1948 in Zika Forest, Uganda (Dick et al., 1952). ZIKV had been long been perceived as a mild illness with fever, rash, arthralgia and conjunctivitis, and may be misdiagnosed as DENV which causes similar symptoms. Sporadic cases of ZIKV infections were reported over half the century before emerging outbreaks in French Polynesia in 2013 and 2014 that were associated with Guillain-Barré syndrome in adults (Cao-Lormeau et al., 2014, Cao-Lormeau et al., 2016, Faye et al., 2014, Oehler et al., 2014). Recent ZIKV outbreaks in South America are associated with severe congenital malformations, most notably microcephaly (Rasmussen et al., 2016) with resulting convulsions, and physical and learning disabilities (Leal et al., 2016a, Leal et al., 2016b, Oliveira Melo et al., 2016). The association of ZIKV infection and microcephaly in mice was recently demonstrated (Cugola et al., 2016, Li et al., 2016, Miner et al., 2016). Despite the rapid spread affecting millions and potentially severe clinical manifestations, there are no licensed anti-ZIKV therapeutics. Multiple potential therapeutic drugs have been identified recently (Adcock et al., 2016, Balasubramanian et al., 2016, Barrows et al., 2016, Deng et al., 2016, Ghezzi et al., 2017, Pascoalino et al., 2016), including FDA-approved drugs such as sofosbuvir and finasteride (Adcock et al., 2016, Barrows et al., 2016, Bullard-Feibelman et al., 2016). ZIKV replication is also significantly inhibited by type I and II interferon (Hamel et al., 2015).

ZIKV has an icosahedral envelope containing a positive-sense, single-stranded RNA genome of approximately 10.7 kb. The open reading frame of ZIKV genome encodes a single polyprotein that is processed by viral proteases and cellular proteases into three structural proteins (capsid, membrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Flaviviruses enter the cell through a clathrin-mediated pathway, and low-pH environment of endosomes trigger membrane fusion of the viral envelope, followed by viral nucleocapsid release into the cytosol (Chu and Ng, 2004, Delvecchio et al., 2016). The attachment of ZIKV is mediated by cellular attachment factors such as DC-SIGN and members of phosphatidylserine kinase family, AXL, Tyro3 and/or TIM-1 (Hamel et al., 2015).

Heparan sulfate (HS) is a negatively-charged linear polysaccharide composed of hexosamine/hexuronic acid repeats. Many pathogens, such as herpes simplex virus (Yura et al., 1992), HIV (Roderiquez et al., 1995), DENV (Chen et al., 1997), Semliki Forest virus (Ferguson et al., 2015), Sindbis virus (Byrnes and Griffin, 1998), chikungunya virus (Silva et al., 2014) and enterovirus A71 (EV-A71) (Tan et al., 2013), are known to utilize cell surface HS as an attachment or entry receptor. HS has been reported as an attachment factor for multiple flaviviruses, including DENV (Chen et al., 1997, Hilgard and Stockert, 2000), JEV (Chen et al., 2010, Chien et al., 2008), tick-borne encephalitis virus (TBEV) (Kozlovskaya et al., 2010, Mandl et al., 2001), and YFV (Germi et al., 2002). It has been demonstrated that virus-HS binding occurs mainly through electrostatic interactions of basic amino acid residues of the virus with negatively-charged sulfate groups (Hu et al., 2011, Knappe et al., 2007, Lortat-Jacob et al., 2002, Tan et al., 2016). To elucidate the role of HS in ZIKV infection, we investigated the inhibitory effect of multiple glycosaminoglycans (GAG) and its analogues (including suramin) against ZIKV infection. Our results highlight a broad-spectrum antiviral agent, suramin, as a potent anti-ZIKV agent with significant antiviral activities at non-cytotoxic concentrations. Our results also showed that, unlike other flaviviruses, ZIKV does not use cell surface HS as an attachment receptor.