Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities.

Chikungunya virus (CHIKV) is an important arboviral infectious agent in tropical and subtropical regions, often causing persistent and debilitating disease. The viral enzyme non-structural protein 4 (nsP4), as RNA-dependent RNA polymerase (RdRP), catalyzes the formation of negative-sense, genomic and subgenomic viral RNAs. Here we report a truncated nsP4 construct that is soluble, stable and purified recombinantly from Escherichia coli. Sequence analyses and homology modelling indicate that all necessary RdRP elements are included. Hydrogen/deuterium exchange with mass spectrometry was used to analyze solvent accessibility and flexibility of subdomains. Fluorophore-conjugated RNA ligands were designed and screened by using fluorescence anisotropy to select a suitable substrate for RdRP assays. Assay trials revealed that nsP4 core domain is conditionally active upon choice of detergent species, and carries out both primed extension and terminal adenylyltransferase activities. The polymerization assay can be further developed to screen for antiviral compounds in vitro.

The crystal structure of the DNA-binding domain of vIRF-1 from the oncogenic KSHV reveals a conserved fold for DNA binding and reinforces its role as a transcription factor.

Kaposi's sarcoma-associated herpesvirus encodes four viral homologues to cellular interferon regulatory factors (IRFs), where the most studied is vIRF-1. Even though vIRF-1 shows sequence homology to the N-terminal DNA-binding domain (DBD) of human IRFs, a specific role for this domain in vIRF-1's function has remained uncertain. To provide insights into the function of the vIRF-1 DBD, we have determined the crystal structure of it in complex with DNA and in its apo-form. Using a thermal stability shift assay (TSSA), we show that the vIRF-1 DBD binds DNA, whereas full-length vIRF-1 does not, suggesting a cis-acting regulatory mechanism in similarity to human IRFs. The complex structure of vIRF-1 DBD reveals interactions with the DNA backbone and the positioning of two arginines for specific recognition in the major grove. A superimposition with human IRF-3 reveals a similar positioning of the two specificity-determining arginines, and additional TSSAs indicate binding of vIRF-1 to an IRF-3 operator consensus sequence. The results from this study, therefore, provide support that vIRF-1 has evolved to bind DNA and plays a role in DNA binding in the context of transcriptional regulation and might act on some of the many operator sequences controlled by human IRF-3.