Molecular Cloning and Characterization of Tha-Virus Proteins

All the sequences were amplified by reverse transcription-polymerase chain reaction (RT-PCR) on RNA extracted from Tha-virus–infected HeLa cells using specific primers. PCR products were inserted into the vector of interest using In-Fusion^TM HD Cloning Kit (Clontech).
Viral P and M sequences were cloned, respectively, with C-terminal and N-terminal FLAG-tags into the pIRES vector (Clontech, PT3266-5) using NheI and XhoI restriction sites. The complete Tha-virus genome was inserted into pSDI-Flash-HH-SC^{37 (link)}, as previously described^{22 (link)}. Plasmids for the protein-complementation assays (PCA) were obtained by cloning M, P, Stat1 and Jak1 sequences into vectors containing the N-terminal (pCMV-KDEL-Glu1) or C-terminal part (pCMV-KDEL-Glu2) of Gaussia luciferase, respectively, using BstXI/SalI and XhoI/SacII restriction sites^{38 (link)}. To study the impact of a third protein in PCAs, plasmids coding for M, P, STAT1 or JAK1 protein without FLAG- or HA-tag were used.
Mutations were introduced into the Tha-genome or P- and/or M-protein plasmids using Change-IT^TM Multiple Mutation Site-Directed Mutagenesis Kit (Afflymetrix) and specific primers, as described previously^{22 (link)}.