The enzyme dimethylallyltryptophan synthase catalyzes the "normal" prenylation of Trp at C-4 in the first step of ergot alkaloid biosynthesis. The Lys174Ala mutant is found to produce a hexahydropyrroloindole alkaloid that is "reverse-prenylated" at C-3 as its major product. This is interpreted as evidence in support of a mechanism that involves an initial "reverse-prenylation" at C-3, followed by a Cope rearrangement and rearomatization.

The prenyltransferase CymD catalyzes the reverse N-prenylation of tryptophan using dimethyllallyl diphosphate (DMAPP) in the biosynthesis of the cyclic peptides cyclomarin and cyclomarazine. The mechanism is of interest since a non-nucleophilic indole nitrogen must be alkylated in this process. Three mechanisms were initially considered that included: A) a direct addition of a carbocation to the nitrogen, B) an addition of a carbocation to C-3 followed by an aza-Cope rearrangement, and C) deprotonation of the indole followed by an SN2′ addition to DMAPP. The use of 4-fluorotryptophan and 6-fluorotryptophan revealed that the reaction kinetics are only modestly affected by these substitutions, consistent with the notion that positive charge does not accumulate on the indole ring during catalysis. When E-3-(fluoromethyl)-2-buten-1-yl diphosphate (E-F-DMAPP) was used in place of DMAPP, the maximal rate was reduced by a factor of 100, consistent with the development of positive charge on the dimethylallyl moiety. Positional isotope exchange (PIX) experiments show that the reaction with Trp proceeds without isotopic scrambling of the label in the starting material [1-18O]-DMAPP. However, in the case of 4-fluorotryptophan, significant isotopic scrambling is observed (vPIX/vrxn = 1.1). This is consistent with a mechanism involving a discrete carbocation intermediate. Finally, a significant solvent kinetic isotope effect of 2.3 was observed in D2O, indicating that a proton transfer step is rate limiting. Taken together, these observations support a mechanism that is a hybrid of mechanisms A and C. Ionization of DMAPP generates a dimethylallyl carbocation, and deprotonation of the indole nitrogen accompanies, or precedes, the nucleophilic attack.

Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae , and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.

RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus. IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

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