Elsevier

Antiviral Research

Volume 112, December 2014, Pages 47-58
Antiviral Research

An adenosine nucleoside analogue NITD008 inhibits EV71 proliferation

https://doi.org/10.1016/j.antiviral.2014.10.009Get rights and content

Highlights

  • Evaluation of 27 nucleoside analogues against EV71.

  • The triphosphorylated NITD008 (ppp-NITD008) directly inhibit the RNA-dependent RNA polymerase activity of EV71.

  • NITD008 presents a significant synergistic anti-EV71 effect with AG7088.

Abstract

Enterovirus 71 (EV71), one of the major causative agents of Hand–Foot–Mouth Disease (HFMD), causes severe pandemics and hundreds of deaths in the Asia-Pacific region annually and is an enormous public health threat. However, effective therapeutic antiviral drugs against EV71 are rare. Nucleoside analogues have been successfully used in the clinic for the treatment of various viral infections. We evaluated a total of 27 nucleoside analogues and discovered that an adenosine nucleoside analogue NITD008, which has been reported to be an antiviral reagent that specifically inhibits flaviviruses, effectively suppressed the propagation of different strains of EV71 in RD, 293T and Vero cells with a relatively high selectivity index. Triphosphorylated NITD008 (ppp-NITD008) functions as a chain terminator to directly inhibit the RNA-dependent RNA polymerase activity of EV71, and it does not affect the EV71 VPg uridylylation process. A significant synergistic anti-EV71 effect of NITD008 with rupintrivir (AG7088) (a protease inhibitor) was documented, supporting the potential combination therapy of NITD008 with other inhibitors for the treatment of EV71 infections.

Introduction

Enterovirus 71 (EV71) is one of the major etiological agents of human Hand–Foot–Mouth Disease (HFMD) in the Asia–Pacific region. In particular, young children and the population with immunodeficiency are more susceptible to EV71 infection. The infection causes severe aseptic meningitis, encephalitis, myocarditis, acute flaccid paralysis, and pulmonary edema, which lead to high fatality rates (Zhang et al., 2010, Cui et al., 2010, Yang et al., 2009, Gautam, 2011, Sun et al., 2012, Zhou et al., 2013).

EV71 is a member of the Enterovirus genus within the Picornaviridae family (Zhou et al., 2013, Chen et al., 2013, Sun et al., 2012). The genome of EV71 contains a 7.5 kb positive-sense single-stranded RNA and encodes a polyprotein that is cleaved by viral proteases to generate four structural proteins (VP1–VP4) required for viral capsid formation and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D) needed for virus replication (McMinn, 2002, Racaniello, 2001, Wu et al., 2010). Among these replicase proteins, the main viral protease (3Cpro) and RNA-dependent RNA polymerase (RdRp) (3Dpol) play critical roles in EV71 replication. After entering the host cell, EV71 produces a nascent polyprotein from its positive-sense genomic RNA, and this polyprotein is subsequently processed into various single or precursor proteins (Pathak et al., 2008, Ypma-Wong et al., 1988, Sun et al., 2014, Yamayoshi et al., 2009). Subsequently, the 3Dpol produces full-length negative-sense RNA by using viral genomic RNA as the template and further generates copies of the viral genomes (Giachetti and Semler, 1991). The RNA replication mainly occurs on the membranous vesicles induced by viral proteins (Dales et al., 1965, Schlegel et al., 1996). In this process, the abundance of 3CD functions to promote replication while repressing the translation event (Gamarnik and Andino, 1998, Lou et al., 2014). Therefore, the effective impairment of the activities of 3Cpro or 3Dpol could be a promising strategy to inhibit the replication of EV71 virus.

Despite the increasing amount of research aimed at identifying antiviral agents against EV71, no direct acting anti-EV71 drugs are currently available in the clinic to combat EV71 infections (Lou et al., 2014). Some compounds have been reported to have inhibitory effects against EV71, including pleconaril, CsA, BPR0Z and GPP3-1 (both target virus entry) (Shang et al., 2013, Zhang et al., 2012, Shia et al., 2002, De Colibus et al., 2014, Qing et al., 2014, Ortiz-Riano et al., 2014), rupintrivir (AG7088) (targets EV71 protease 3Cpro (Dragovich et al., 1999), the peptide LVLQTM (targets 2Apro (Falah et al., 2012), DTriP-22 and aurintricarboxylic acid (target polymerase 3Dpol (Chen et al., 2009, Urbinati et al., 2008) and several natural products such as lycorine and raoulic acid (Choi et al., 2009, Liu et al., 2011).

Nucleoside or nucleotide analogues are known to inhibit viral genome replication by acting as chain terminators that are incorporated into the DNA/RNA strands to block the replication of DNA/RNA or that affect host cell pathways (Graci and Cameron, 2006, Yin et al., 2009). Nucleoside or nucleotide analogues have been well accepted as therapeutic agents and play a dominant role in the treatment of a number of viral infections, e.g., lamivudine and tenofovir for anti-HBV, zidovudine and stavudine for anti-HIV (Lou et al., 2014). In a previous report, ribavirin, a nucleoside analogue, was revealed to have a 50% effective concentration (EC50) at 89.23–178.42 μg/ml (360–730 μM) against EV71 in a cell-based assay (Zhang et al., 2012, Urbinati et al., 2008). The antiviral activity of ribavirin is mediated by several non-mutually exclusive mechanisms including inhibition of the cellular inosine-monophosphate dehydrogenase and viral mutagenesis (Graci and Cameron, 2006). In the present work, we screened a collection of nucleoside analogues and found NITD008 (compound 11), which was reported to be an antiviral reagent that specifically inhibits flaviviruses (Yin et al., 2009), effectively inhibited the replication of various strains of EV71 in human (RD and 293T) and monkey (Vero) cell lines with a best EC50 value of 0.1 μM. Triphosphorylated NITD008 (ppp-NITD008) functions as a chain terminator to directly inhibit the RNA-dependent RNA polymerase activity of EV71 but does not affect the initiation of replication. We also found that the combination of NITD008 and AG7088 exhibited a strong synergistic anti-EV71 effect, providing great potential to treat EV71 infection and to circumvent some of the limitations of monotherapy.

Section snippets

Viruses, cell lines and antibody

The African green monkey kidney (Vero) cells (ATCC, accession No. CCL-81), human rhabdomyosarcoma (RD) cells and human embryonic kidney 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37 °C in a 5% CO2 humidified incubator.

EV71 (strain SK-EV006) with GFP (EV71-GFP) was provided kindly by Prof. Satoshi Koike for the initial phenotype screening. A single round pseudotype EV71 reporter virus system (EV71(FY)-Luc

NITD008 is a potent inhibitor against EV71 infection

Our aim in this work is finding a nucleoside analogue that can inhibit the replication of EV71. Quick phenotype screening was performed using a total of 27 nucleoside analogues that were initially assayed for their antiviral activity against EV71 (Table 1) by using RD cells infected with EV71 strain SK-EV006 with GFP (Oberg, 2006) that expressed upon infection. RD cells were infected with virus (MOI of 1) for 5 min and immediately treated with various concentrations of different compounds

Discussion

Currently, there are three recognized types of polymerase inhibitors, including substrate analogs (nucleoside and nucleotide analogs), product analogs (pyrophosphate analogs) and allosteric inhibitors (Lou et al., 2014). The nucleoside or nucleotide analogues occupy the dominant position of all antiviral reagents in either clinical use or under development (Lou et al., 2014). It should be noted that PSI-7977 is the exclusive RNA polymerase inhibitor licensed as a drug against HCV infection, but

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant Nos. 81322023, 31370733, 31170678, 31100208, 31000332 and 21202087), the National Basic Research Program of China (973 program, Grant Nos. 2013CB911100 and 2014CB542800), the Fundamental Research Funds for the Central Universities (Grant No. 65124002), the Specialized Research Fund for the Doctoral Program of Higher Education Ministry of Education of China (Grant No. 20120031120049), the Tianjin Science and

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    These authors contributed equally to this work.

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    These authors are co-senior authors of this work.

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