Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor

Abstract

Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15-25, 2013, doi:10.1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/ release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses. Dengue virus (DV) belongs to the genus Flavivirus, a large group of emerging pathogens capable of causing severe human diseases. Among these, DV represents the most prevalent mosquito-borne human-pathogenic virus worldwide, comprising 4 serotypes that are transmitted mainly by infected Aedes mosquitoes during a blood meal. DV infections can lead to a wide range of clinical manifestations, ranging from asymptomatic infections to life-threatening dengue hemorrhagic fever and shock syndrome. A recent study estimated around 390 million DV infections each year, resulting in approximately 100 million symptomatic cases and around 25,000 deaths (1). Despite intense efforts and growing public interest, no licensed antiviral drug against DV infection is available, and the most advanced DV vaccine candidate did not meet expectations in a recent large clinical trial (2).DV has a single-stranded RNA genome of positive polarity that codes for a polyprotein, which is co-and posttranslationally processed into three structural proteins (capsid, prM, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3). The virus enters mammalian cells via receptor-mediated endocytosis. In the endosomal compartment, the low pH induces a conformational change in the envelope (E) protein, triggering membrane fusion and nucleocapsid release into the cytoplasm (4, 5). Disassembly of the nucleocapsid occur...

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

“…This genetic complementation results in the production of pseudoinfectious virus-like particles capable of only a single round of infection, referred to herein as reporter virus particles (RVPs). RVPs have been used extensively to study flavivirus biology (43)(44)(45)(46)(47)(48) and virus-antibody interactions (35,36,(49)(50)(51) and as tools to screen antiviral compounds (19,52,53). The mechanism of flavivirus RNA packaging is not well understood and appears to be relatively nonselective.…”

Fexaramine as an entry blocker for feline caliciviruses

“…Interestingly, investigation on the mode of FCV inhibition by fexaramine by various experiments including the time-of-addition studies and analysis of viral resistance showed that, in contrast with inhibition of rotavirus , fexaramine acts as an entry inhibitor of FCV. Virus entry inhibitors have been developed or reported for enveloped viruses including human immunodeficiency virus (HIV) (Haqqani and Tilton, 2013;Woollard and Kanmogne, 2015), influenza virus (Palese and Shaw, 2007), Measles virus (Plemper et al, 2005) and Dengue virus (Scaturro et al, 2014). Virus entry inhibitors have also been reported for non-enveloped viruses including picornaviruses including poliovirus (Thys et al, 2008), enterovirus (Buontempo et al, 1997;Ho et al, 2016;Ma et al, 2017;Torres-Torres et al, 2015) and rhinoviruses Kim et al, 2017;Tijsma et al, 2014).…”

Properties and Functions of the Dengue Virus Capsid Protein

“…A recent high-throughput screen identified a small molecule (ST-148) that potently inhibits replication of all four DENV serotypes in vitro by targeting the capsid protein (130). ST-148 has been proposed to enhance capsid self-interaction, likely perturbing viral assembly and disassembly (91). Interestingly, despite the presence of viral variants with resistance to ST-148 in the population, selection for resistant viruses was not observed (131).…”