Deep mutational scanning reveals the functional constraints and evolutionary potential of the influenza A virus PB1 protein.

IMPORTANCE: The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus' natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.

Mutual information networks reveal evolutionary relationships within the influenza A virus polymerase.

The influenza A virus (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 (polymerase basic protein 2, polymerase basic protein 1, and polymerase acidic protein). 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 (wMI) metric and demonstrate that wMI outperforms raw MI through simulations using a well-sampled severe acute respiratory syndrome coronavirus 2 (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 hemagglutinin (HA) in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitch-hiking 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.

Mutual information networks reveal evolutionary relationships within the influenza A virus polymerase.

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.

ELAVL1 primarily couples mRNA stability with the 3' UTRs of interferon-stimulated genes.

Upon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3' UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries.

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes.

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 crosslinked 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 crosslinking 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 crosslinked 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.

Discovery of Widespread Host Protein Interactions with the Pre-replicated Genome of CHIKV Using VIR-CLASP.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.