The clinical efficacy of sulfa drugs as antimalarials has declined owing to the evolution of resistance in Plasmodium falciparum (Pf) malaria parasites. In order to understand the basis of this resistance and to design more effective antimalarials, we have solved 13 structures of the bifunctional enzyme 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (HPPK)–dihydropteroate synthase (DHPS) from wild‐type (WT) P. falciparum and sulfa‐resistant mutants, both as apoenzyme and as complexes with pteroate (PTA) and sulfa derivatives. The structures of these complexes show that PTA, which effectively inhibits both the WT and mutants, stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants. Kinetic data show that the mutants have enhanced enzyme activity compared with the WT. These PfDHPS structural insights are critical for the design of novel, substrate envelope–compliant DHPS inhibitors that are less vulnerable to resistance mutations.DatabasesThe data reported in this paper have been deposited in the Protein Data Bank, www.wwpdb.org. PDB ID codes: 6JWQ for apoWT; 6JWR, 6JWS, and 6JWT for PTA complexes of WT, A437G (3D7), and V1/S; 6JWU, 6JWV, and 6JWW for STZ‐DHP complexes of WT, 3D7, and V1/S; 6JWX, 6JWY, and 6JWZ for SDX‐DHP complexes of WT, 3D7, and W2; 6KCK, 6KCL, and 6KCM for Pterin/pHBA complexes of WT, TN1, and W2.

The ORF3 accessory protein has been shown to impede reverse genetics of cell-culture-adapted porcine epidemic diarrhea virus (PEDV). Its absence or truncated variants are also associated with viral attenuation in vivo. Here, three ORF3 variants (ORF3, ORF3 and ORF3) and their truncated counterparts were investigated for their regulatory role in recovery of cell-adapted PEDV in vitro. We demonstrate that ORF3, but not the truncated form, can inhibit recovery of reverse-genetics-derived PEDV when expressed in trans. When testing with other RNA viruses, ORF3 was found to inhibit rescue of porcine respiratory and reproductive syndrome virus (PRRSV), but not of influenza virus. Interestingly, results from mutagenesis of ORF3 suggest that F81 and M167 are responsible for impairing PEDV rescue in vitro. By changing specific residues of ORF3, the recombinant PEDV bearing the modified ORF3 can be productively propagated in VeroE6-APN cells. These results may provide mechanistic insights into ORF3-mediated inhibition of PEDV replication in new host cells.

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.

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