Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes capping of genomic and subgenomic RNAs. The capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming GTP; (ii) guanylylation of the enzyme to form a GMP-nsP1adduct; (iii) transfer of GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions.Copyright © 2020. Published by Elsevier B.V.