Figure 10. Effects of C003 and C052 on BTV VP6 protein expression and LC3-I/LC3-II conversion were analyzed in sample collected at different h.p.i. Cells were infected with BTV at MOI of 0.01, and treated with C003 (10 mM) or C052 (2.5 mM). VP6 expressions and LC3-I/LC3-II conversion in these samples were determined using the western blot. doi:10.1371/journal.pone.0043341.g010 autophagy in BTV-infected cells, with and without virostatic compound treatment. When infected at MOI of 0.01, autophagy was not activated at 24 h post BTV infection as the level of LC3-II showed no difference in sample with or without C003/C052 treatment. However, at 48 h.p.i., there was a turnover of LC3-I to LC3-II in BTV infected cells without C003/C052 treatment, with the LC3-I/LC3-II ratio at 0.05 which indicating most of LC3-I was cleaved into LC3-II (Fig. 10). The turnover of LC3-I to LC3- II was at a much lower level when C003 at 10mM or C052 at 2.5 mM was added to the BTV-infected cells, with the ratio of LC3-I/LC3-II at 0.83 and 0.71 respectively (Fig. 10). This result suggested that C003/C052 might act on interfering with BTV induced autophagy, consequently, inhibiting BTV-induced CPE. Discussion As a result of its economic significance and as a model system, BTV has been the subject of extensive molecular, genetic and structural studies, and now represents one of the most well characterized viruses [7,10,11,15]. Much of the efforts were focused on developing vaccines against BTV, including the attenuated BTV and VLPs [18,49,50]. In contrast, it remains under-investigated to utilize the knowledge from the molecular and virological studies for antiviral drug discovery. Here, we reported the characterization of a novel class of compound as virostatic agents against BTV. These compounds, i.e. the thiophene derivatives, have been identified as active compounds in various assays, including as an enhancer of survival motor neuron protein splice variant expression; an activator for alpha- synuclein 59UTR - 59-UTR binding, and for STAT1 activation [51]. It is plausible that the aminothiophenecarboxylic acid derivative–C003 was identified as a novel compound against BTV, with potent virostatic efficacy, low cytotoxicity and high SI. More interestingly, the preliminary SAR analysis of C003 leads to an improved virostatic compound–C052, via de novo synthesis. Our studies suggest that using C003/C052 as lead compounds, it is possible to develop novel and potent antivirals against BTV via chemical modification, while maintain its low toxicity and high selectivity. For the majority of RNA viruses, the induction of apoptosis, which facilitates the virus dissemination, is an essential step in the Virostatic Agents against Bluetongue Virus late stage of viral life-cycle. Interfering the host apoptotic machinery, especially inhibiting the virus induced apoptosis, will disrupt the successful viral life-cycle via blocking virus release [52,53,54]. Thus, host apoptotic response post virus infection is an attractive target for antiviral drug development because it is a common and indispensible late step for a productive viral life- cycle of many, if not all, viruses. Previous studies have shown that BTV infection triggers apoptosis via both intrinsic and extrinsic pathways [1,37,55]. The induction of apoptosis is involved in BTV-induced pathogenesis in vivo [56,57]. Our mechanism of action studies indicated that C003 and C052 were active by protecting cells from BTV-induced apoptosis. The inhibition of caspsse-3/7 activation indicated the targets should be in the upstream of the cascade of caspase activation. However, when BSR cells were treated with pan-caspase inhibitor, including Z- VAD-FMK, cells were not protected from BTV-induced apopto- sis. BTV-induced CPE was observed, and BTV replication was not interrupted (results not shown). This suggested that while apoptosis/CPE was inhibit by C003/C052, the mechanism of action of these virostatic compounds might not directly related to BTV-induced apoptotic pathways, rather as a results of inhibiting a late step in viral life-cycle or host machineries inducing upstream apoptotic signals. Interestingly, our results showed that the host autophagy machinery/pathway was inhibited by C003 and/or C052, as LC3-II, the only reliable autophagy marker, was down-regulated when BTV-infected cells were treated with these compounds (Fig. 10). Autophagy is a highly conserved process in eukaryotes in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to the degradative organelle, the lysosome/vacuole, for breakdown and eventual recycling of the resulting macromolecules [44,47,58]. Autophagy is activated as an adaptive response to a variety of extracellular and intracellular stimuli, including nutrient depriva- tion, hormonal or therapeutic treatment, aggregated and mis- folded proteins, damaged organelles and microbial infection [58,59,60]. Although the autophagy pathway might be a host defense mechanism, different viruses have developed various strategies to counteract these antiviral mechanisms, and/or to utilize the autophagy machinery as proviral host factors favoring viral replication [46,61]. Recently, a growing list of viruses has been shown to be targeted for autophagic degradation [62]. For instance, autophagy is actively involved in influenza A virus replication by triggering autophagosome formation, increasing the level of LC3-II and enhanced autophagic flux [45]. Dengue virus can also activate autophagic machinery that is favorable for viral replication [48,63], and autophagosomes act as a site for translation and replication of dengue virus-2 and that its entry and replication are linked through an ongoing association with membranes of an endosomal–autophagosomal lineage [64,65]. Similarly, induction of autophagy pathways can enhance the replication of poliovirus and coronavirus [66,67]. During the infection of dsRNA virus rotavirus, nonstructural protein 4 (NSP4) colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication [68,69]. Our finding showed that C003 and C052 interacted with autophagy machinery, which may lead to a new research avenue to develop novel antivirals intervene virus- autophagy interactions. While virus entry and virus replication have been targets for extensive antiviral drug discovery and development, egress/ dissemination of a newly synthesized progeny virus, equally important in relation to successful viral infection, receives much PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43341