FRET studies examining the interaction of hepatitis C NS3 helicase and human DExD/H box helicases
Authors: Corby Josie , Biener Gabriel , Raicu Valerica , Frick David 
Published: November 2023

The innate immune response to viral infection is the intracellular first line of defense against invading pathogens. One strategy that viruses, such as hepatitis C virus (HCV), use to establish a chronic infection is to short circuit this innate immune response. HCV encodes a multi‐functional non‐structural protein 3 (NS3), which is a protease covalently tethered to a C‐terminal helicase. One role of the NS3 protease in innate immune response evasion is to cleave mitochondrial anti‐viral signaling protein (MAVS). The function of the NS3 helicase is essential for replication but it is unclear why it is covalently linked to the protease.

We propose that HCV NS3 helicase works to evade the innate immune response by binding to pathogen associated molecular patterns (PAMPs) in the HCV genome and interacting with the RIG‐I like receptor LGP2, to localize the NS3 protease near MAVs for cleavage.

In order to study the interaction between human and viral helicases we used a process called Förster resonant energy transfer (FRET) where an excited fluorescent molecule (donor) transfers its energy to a nearby fluorescent molecule of a different type (acceptor) which in turn emits a red shifted fluorescent light. This process occurs in distances smaller than 10 nm between the donor and acceptor which indicates obvious interaction.

We generated two sets of plasmids encoding donor (mCFP) and acceptor (mYFP) fused to viral and human helicases respectively. The donor plasmid set encodes recombinant proteins in which all or part of HCV NS3 and its cofactor NS4A were fused to a mCFP fluorescent protein. The first expresses only the NS3 helicase domain (mCFP‐NS3h) fused to mCFP. In the second, mCFP was fused with full‐length NS3 (mCFP‐NS3). In the third, mCFP was fused to NS3 and only the NS3 cofactor domain of NS4A (lacking the membrane anchor and C‐terminus, i.e. mCFP‐NS3‐4Acd), and in the fourth, mCFP was fused to NS3 and full length NS4A (mCFP‐NS3‐NS4A). The acceptor plasmid set encodes recombinant mYFP fused to DDX1, DDX3, DDX5, and DHX 58 (LGP2). HEK293T cells were co‐transfected with each donor plasmid and acceptor plasmid. The live cells were imaged using an optical micro‐spectroscope (OptiMis) which consists of a two photon excitation scanning optical microscope with high spectral resolution. In a separate set of experiments each of the donor plasmids were co‐transfected with a plasmid expressing RFP fused to MAVs and a nuclear localization signal.

The fluorescently labeled recombinant NS3 proteins were visualized in real‐time in HEK293T cells. The RFP MAVs re‐localized to the nucleus only when it was co‐transfected with plasmids that encoded NS4A, in addition to the NS3 protease. FRET was observed between all recombinant HCV NS3 proteins and LGP2, but not between any recombinant HCV NS3 proteins and closely related DDX 1, DDX3, and DDX5. Our data indicates that the NS3 fusion proteins are biologically active and that NS3 interacts with LGP2 via its helicase domain.