Antiviral activity against enterovirus 71 of sulfated rhamnan isolated from the green alga Monostroma latissimum

Highlights

• Sulfated polysaccharide PML was prepared from the green alga Monostroma latissimum.

• PML effectively blocked EV71infection in vitro and showed excellent anti-EV71 activity in vivo.

• PML could strongly bind to VP1 protein of EV71 with evident dose dependence.

• PML inhibit EV71 infection through targeting cellular EGFR/PI3K/Akt pathway.

• PML could be a novel antiviral agent for therapy and prophylaxis of EV71 infection.

Abstract

Polysaccharide from Monostroma latissimum PML is a sulfated rhamnan, which consists of →3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→ residues with partial branches and sulfate groups at C-2 of →3)-α-L-Rhap-(1→ and/or C-3 of →2)-α-L-Rhap-(1→. The anti-enterovirus 71 (EV71) activity in vitro of PML was assessed by cytopathic effect inhibition and plaque reduction assays, and the results showed that PML was non-cytotoxic and significantly inhibited EV71 infection. The mechanism analysis of anti-EV71 activity demonstrated that PML largely inhibited viral replication before or during viral adsorption, mainly by targeting the capsid protein VP1. PML may also inhibit some early steps of infection after viral adsorption by modulating signaling through the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Moreover, PML markedly improved survival and decreased viral titers in EV71-infected mice. The investigation revealed that PML has potential as a novel anti-EV71 agent targeting the viral capsid protein as well as cellular EGFR/PI3K/Akt pathway.

Introduction

Enterovirus 71 (EV71) is a non-enveloped, positive-sense single-stranded RNA virus, which is one of the major causative agents of hand, foot and mouth disease in infants and young children. In some individuals, EV71 can infect the central nervous system and cause severe and life-threatening neurological disorders, including aseptic meningitis acute flaccid paralysis, myoclonus with autonomic dysfunction, encephalitis, and cardiac failure (Ooi, Wong, Lewthwaite, Cardosa, & Solomon, 2010; Tsai et al., 2012). According to the National Health and Family Planning Commission of the People’s Republic of China, EV71 caused at least 17,717,819 infections and 3,509 deaths from 2009 to 2017 in China. Only a few vaccines and antiviral drugs have been approved for prophylaxis or treatment of EV71 infection (Pourianfar & Grollo, 2015). Hence, development of efficacious and non-toxic anti-EV71 drugs is urgently needed.

A single enterovirus virion is typically composed of 60 copies of capsids, each of which is made up of four capsid proteins: VP1, VP2, VP3 and VP4 (Shia et al., 2002). It has been reported that VP1 might contain receptor-binding sites, and that it might determine cell tropism and regulate virus maturation. Thus, VP1 inhibitors have been proposed as candidate for antiviral against EV71. Moreover, the cellular PI3K/Akt signaling pathway plays important roles in cell survival, apoptosis, proliferation, migration and differentiation, as well as in metabolic regulation. This pathway has also been reported to be associated with virus uptake and virus induced autophagy (Diehl & Schaal, 2013; Lin et al., 2017). Thus, inhibitors of viral VP1 protein and cellular PI3K/Akt pathway may be used alone or in combination with other drugs to block both infection and replication of EV71.

Sulfated polysaccharides from green algae often possess potential antiviral activities (Ngo & Kim, 2013; Shi et al., 2017). An acidic polysaccharide purified from the green alga Coccomyxa gloeobotrydiformi was reported to exhibit the anti-influenza virus activity through preventing interactions between virus and host cells (Komatsu, Kido, Sugiyama, & Yokochi, 2013). A sulfated polysaccharide from Enteromorpha compressa showed significant anti-herpes simplex virus (HSV)-1 activity primarily by affecting viral replication (Lopes et al., 2017). Antiviral activities of sulfated polysaccharides from Monostroma sp. were also reported (Lee, Hayashi, Hayashi, Sankawa, & Maeda, 1999; Lee, Koizumi, Hayashi, & Hayashi, 2010). Rhamnan sulfate from Monostroma latissimum had potent antiviral activities against HSV-1 and human immunodeficiency virus type 1 (HIV-1) (Lee et al., 1999). The sulfated polysaccharide from M. nitidum showed potent inhibition of HSV-2 adsorption and penetration (Lee et al., 2010). The sulfated polysaccharides from green algae have potential as novel antiviral agents.

The green alga M. latissimum grows in upper part intertidal zone and widely cultivated as an edible alga (Lee, Yamagaki, Maeda, & Nakanishi, 1998; Li et al., 2011). Our previous study indicated that the polysaccharide, PML, isolated from M. latissimum was a novel sulfated rhamnan and possessed strong anticoagulant activity (Li et al., 2011). The purpose of this study was to investigate the inhibitory effects and mechanisms of PML against EV71. PML was found to be able to effectively inhibit EV71 activity in vitro with low cytotoxicity, markedly reduce viral loads and prevent death of EV71-infected mice. The anti-EV71 activity of PML might involve inhibition of EV71 invasion and replication by targeting the viral capsid protein and cellular EGFR/PI3K/Akt pathway. The results of our analyses are presented in this paper.