Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model q Abhay P.S. Rathore a , Prasad N. Paradkar a,1 , Satoru Watanabe a , Kah Hin Tan a , Cynthia Sung a , John E. Connolly b , Jenny Low c , Eng Eong Ooi a , Subhash G. Vasudevan a,⇑ a Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore b Program in Translational Immunology, Singapore Immunology Network (SIgN), 8A Biomedical Grove, Immunos Building, Level 4, Singapore 138648, Singapore c Department of Infectious Diseases, Singapore General Hospital, Singapore 169608, Singapore article info Article history: Received 7 August 2011 Revised 5 September 2011 Accepted 6 October 2011 Available online 12 October 2011 Keywords: Dengue virus Celgosivir a-Glucosidase inhibitor Dengue NS1 protein misfolding AG129 mouse model for dengue infection abstract Dengue virus (DENV) infections continue to spread aggressively around the world. Here we demonstrate that celgosivir (6-O-butanoyl castanospermine), strongly inhibits all four DENV serotypes. We show by fluorescence microscopy that the antiviral mechanism of celgosivir, is in part, due to misfolding and accu- mulation of DENV non-structural protein 1 (NS1) in the endoplasmic reticulum. Moreover, celgosivir modulates the host’s unfolded protein response (UPR) for its antiviral action. Significantly, celgosivir is effective in lethal challenge mouse models that recapitulate primary or secondary antibody-dependent enhanced DENV infection. Celgosivir treated mice showed enhanced survival, reduced viremia and robust immune response, as reflected by serum cytokine analysis. Importantly, survival increased even after treatment was delayed till 2 days post-infection. Together the present study suggests that celgosivir, which has been clinically determined to be safe in humans, may be a valuable candidate for clinical test- ing in dengue patients. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Dengue fever (DF) is the most prevalent mosquito-borne viral infection in humans and is caused by DENV, a member of the Flaviviridae family. Infection of humans by any of the four DENV serotypes leads to an incubation period ranging from 3 to 14 days (average 4–7 days) followed by the rapid onset of clinical manifes- tations of DF (Guzman et al., 2010; Halstead, 2007). While DF is usually self-limiting, some patients progress to life-threatening se- vere diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The phenomenon that non-neutralizing heterotypic antibodies can cause antibody-dependent enhance- ment (ADE), together with the increasing prevalence of DENV infections around the world, raises the probability of increasing cases of severe DENV disease (Sabin, 1952; Thein et al., 1997; Whitehorn and Farrar, 2010). Despite the observed association be- tween DHF/DSS and higher viremia compared to DF (Vaughn et al., 2000), no antiviral treatment exists. There is, thus, a global urgency to develop drugs that can reduce the burden of DENV morbidity and mortality (Keller et al., 2006). The plus-strand RNA genome of DENV is translated as a single polyprotein precursor which is processed by host and virus-en- coded proteinases to produce three structural proteins (Capsid, prM, Envelop) and seven non-structural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Chambers et al., 1990). The virus assembly occurs in the endoplasmic reticulum (ER), where hetero- dimers of pre-membrane protein (prM) and envelope (E) localize to the luminal side of the ER to form an immature particle (Chambers et al., 1990; Zhang et al., 2003). Previous studies have shown that N-linked glycosylation of prM and E proteins are required for the proper assembly and release of infectious DENV particles (Coura- geot et al., 2000). Celgosivir is a water soluble oral pro-drug of the natural alka- loid castanospermine (Cast), derived from the Moreton Bay chest- nut tree (Castanospermum australae)(Molyneux et al., 1986). It readily crosses cell membranes and is rapidly converted to Cast (Kang, 1996). The compound inhibits the catalytic activity of ER resident enzymes, a-glucosidase I and II, which play a critical role in the proper folding of N-glycosylated glycoproteins. Treatment with Cast may affect the folding of some viral proteins by prevent- ing the removal of the terminal glucose residue from N-linked gly- cans (Taylor et al., 1991). Lack of modification of the high mannose 0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2011.10.002 q Parts of the information have been presented at the International Conference on Antivirals for Neglected, Emerging Viruses (ICAV-9) in Lubeck, Germany, Germany (October 10–13, 2010), the 5th Ditan International Conference on Infectious Diseases held in Beijing, China (July 14–17, 2011). ⇑ Corresponding author. Tel.: +65 6516 6718; fax: +65 6221 2529. E-mail address: subhash.vasudevan@duke-nus.edu.sg (S.G. Vasudevan). 1 Current address: CSIRO Livestock Industries, Australian Animal Health Labora- tory, Private Bag 24, Geelong, Victoria 3220, Australia. Antiviral Research 92 (2011) 453–460 Contents lists available at SciVerse ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral