Understanding the dynamics of host innate immune responses against a pathogen marks the first step toward developing intervention strategies against the pathogen. The cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been shown to be the major innate immune sensor for hepatitis E virus (HEV). Here, we show that HEV capsid protein (ORF2), a 660 amino acid long protein, interferes with the RIG-I signaling. Interestingly, only the full length ORF2 protein but not the 112-608 ORF2 protein inhibited RIG-I dependent interferon response. Both synthetic agonist and virus induced RIG-I activation was modulated by ORF2. Interference of interferon response was confirmed by reporter assays involving different interferon inducible promoters, qRT PCR, ELISA, and immunofluorescence microscopy. Neither glycosylation nor dimerization of the ORF2 protein had any effect on the observed inhibition. Further analyses revealed that the ORF2 protein antagonized Toll-like receptor (TLR) pathways as well. ORF2 inhibited signaling by RIG-I and TLR adapters, IPS-1, MyD88, and TRIF but was unable to inhibit activation by ectopically expressed IRF3 suggesting that it may be acting at a site upstream of IRF3 and downstream of adapter proteins. Our data uncover a new mechanism by which HEV may interfere with the host antiviral signaling.

Cytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.

Lipid‐based palmitoylation is a post‐translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl‐transferase enzymes containing the conserved DHHC‐CRD (DHHC motif within a cysteine‐rich domain) which can serve as a druggable target. However, the paucity of high‐throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM‐null system, to enforce ectopic expression of palmitoyl acyl‐transferase in order to study Plasmodium‐specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium‐specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl‐transferase activity via acyl‐biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC‐expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2‐bromopalmitate (2‐BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high‐throughput screening platform.

Hepatitis C virus (HCV) is a global pathogen and infects more than 185 million individuals worldwide. Although recent development of direct acting antivirals (DAA) has shown promise in HCV therapy, there is an urgent need for the development of more affordable treatment options. We initiated this study to identify novel inhibitors of HCV through screening of compounds from the National Cancer Institute (NCI) diversity dataset. Using cell-based assays, we identified NSC-320218 as a potent inhibitor against HCV with an EC50 of 2.5 μM and CC50 of 75 μM. The compound inhibited RNA dependent RNA polymerase (RdRp) activity of all six major HCV genotypes indicating a pan-genotypic effect. Limited structure-function analysis suggested that the entire molecule is necessary for the observed antiviral activity. However, the compound failed to inhibit HCV NS5B activity in vitro, suggesting that it may not be directly acting on the NS5B protein but could be interacting with a host protein. Importantly, the antiviral compound also inhibited dengue virus and hepatitis E virus replication in hepatocytes. Thus, our study has identified a broad-spectrum antiviral therapeutic agent against multiple viral infections.