Highlights d VIR-CLASP identifies host protein interactions with incoming RNA viral genomes d VIR-CLASP can reveal the early interactomes of seven different viral families d The CHIKV genome binds to distinct proteins under different conditions and times d (Non)canonical RBPs like FASN, IFI16, and YTHDF1 uniquely regulate CHIKV replication

ELAVL1 primarily couples mRNA stability with the 3 0 UTRs of interferon-stimulated genes Graphical abstract Highlights d ELAVL1 changes its mRNA binding distribution during innate immunity d ELAVL1 stabilizes the mRNAs of interferon-stimulated genes (ISGs) d RNA half-lives of ISG targets decrease in the absence of ELAVL1

Upon detection of a pathogen, the innate immune system triggers signaling events leading to the transcription of mRNAs that encode for pro-inflammatory and anti-microbial effectors. RNA-binding proteins (RBPs) interact with these functionally critical mRNAs and temporally regulate their fates at the post-transcriptional level. One such RBP is ELAVL1, which is known to bind to introns and 3'UTRs. While significant progress has been made in understanding how ELAVL1 regulates mRNAs, how its target repertoire and binding affinity changes within an immunological context remains poorly understood. Here, we overlap four distinct high-throughput approaches to define its cell-type and context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 overwhelmingly binds to intronic sites in a naive state, but during an innate immune response, ELAVL1 targets the 3'UTR - binding both previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate the pathways involved in pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to best understand their post-transcriptional regulatory roles within specific biological circuitries.

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU labeled viral genomes and interacting proteins from host or virus. The cross-linked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so cross-linking events specifically occur between proteins and pre replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and cross-linked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ~7 days and VIR-CLASP takes 1 day.

The influenza A (IAV) RNA polymerase is an essential driver of IAV evolution. Mutations that the polymerase introduces into viral genome segments during replication are the ultimate source of genetic variation, including within the three subunits of the IAV polymerase (PB2, PB1, and PA). Evolutionary analysis of the IAV polymerase is complicated, because changes in mutation rate, replication speed, and drug resistance involve epistatic interactions among its subunits. In order to study the evolution of the human seasonal H3N2 polymerase since the 1968 pandemic, we identified pairwise evolutionary relationships among ~7000 H3N2 polymerase sequences using mutual information (MI), which measures the information gained about the identity of one residue when a second residue is known. To account for uneven sampling of viral sequences over time, we developed a weighted MI metric (wMI) and demonstrate that wMI outperforms raw MI through simulations using a well-sampled SARS-CoV-2 dataset. We then constructed wMI networks of the H3N2 polymerase to extend the inherently pairwise wMI statistic to encompass relationships among larger groups of residues. We included HA in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitchhiking on antigenic changes in HA. The wMI networks reveal coevolutionary relationships among residues with roles in replication and encapsidation. Inclusion of HA highlighted polymerase-only subgraphs containing residues with roles in the enzymatic functions of the polymerase and host adaptability. This work provides insight into the factors that drive and constrain the rapid evolution of influenza viruses.