Applying Reverse Genetics to Study Measles Virus Interactions with the Host.

The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.