Biology subjects

Russell, A. B.

Publications and source records attributed to Russell, A. B..

10 recordsLinked to original sources

bioRxiv2026

Disruption of the polyuracil tract can greatly enhance immunostimulatory properties of influenza delVGs

Non-standard viral genomes (nsVGs) are replication-incompetent genomes within viral populations whose presence has been associated with an enhanced immune response. This association has garnered interest in their use as potential antivirals, vaccine adjuvants, or general immune-modulators. In influenza A virus (IAV), the predominant nsVG is a large deletion in one of the three polymerase segments, which results in deletion-containing viral genomes (delVGs). Work from our group has found that individual delVGs can be recovered that are capable of stimulating high levels of interferon, however we and others have not previously found any consistent means of predicting or modifying determinants of this stimulation. Using high-throughput genetic approaches, we find that specific mutations disrupting the polyuracil tract in IAV delVGs can enhance interferon production by up to two orders of magnitude. Such variants can also associate with improved rescue in artificial systems engineered to exclude infectious virus, and can greatly suppress the growth of replication-competent virus. These findings indicate the polyuracil tract is a key, and unexpected, target for improvement of immunostimulatory delVGs derived from IAV.

microbiology↗

bioRxiv2024

Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

Transcription of interferons upon viral infection is critical for cell-intrinsic innate immunity. This process is influenced by many host and viral factors. To identify host factors that modulate interferon induction within cells infected by influenza A virus, we developed CRISPR with Transcriptional Readout (CRITR-seq). CRITR-seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor (NELF) complex increases both flu transcription and interferon expression. We find that the process of flu transcription, both in the presence and absence of viral replication, is a key contributor to interferon induction. Taken together, our findings highlight innate immune ligand concentration as a limiting factor in triggering an interferon response, identify NELF as an important interface with the flu life cycle, and validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression. Significance StatementNearly every cell in the human body has the ability to detect and respond to viral infection by producing interferons. The timing and magnitude of the interferon response impacts the course of disease, and both hosts and viruses have evolved mechanisms to regulate interferon induction within infected cells. It has previously been challenging to comprehensively search for regulators of interferon expression using selection-based screens. Here we developed a CRISPR screening strategy to measure the effects of gene edits on transcription at a promoter of interest. Applying this method to study interferon transcription during influenza infection, we identified an interface between human and influenza transcription machinery that modulates the viral life cycle and influences the interferon response.

molecular biology↗

bioRxiv2024

Influenza A virus transcription generates capped cRNAs that activate RIG-I

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable in vitro and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

microbiology↗

bioRxiv2024

Efficient genome replication in influenza A virus requires NS2 and sequence beyond the canonical promoter

Influenza A virus encodes promoters in both the sense and antisense orientations. These support the generation of new genomes, antigenomes, and mRNA transcripts. Using minimal replication assays--transfections with viral polymerase, nucleoprotein, and a genomic template--the influenza promoter sequences were identified as 13nt at the 5 end of the viral genomic RNA (U13) and 12nt at the 3 end (U12). Other than the fourth 3 nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules that comprise the segmented influenza genome. Despite possessing identical promoters, individual segments can exhibit different transcriptional dynamics during infection. However flu promoter sequences were defined in experiments without influenza NS2, a protein which modulates transcription and replication differentially between genomic segments. This suggests that the identity of the "complete" promoter may depend on NS2. Here we assess how internal sequences of two genomic segments, HA and PB1, may contribute to NS2-dependent replication as well as map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequence, providing a mechanism for the divergent replication and transcription dynamics across the influenza A virus genome.

microbiology↗

bioRxiv2023

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

microbiology↗

bioRxiv2022

Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection. Author summaryThe production of interferons in response to viral threat is a potent barrier to successful infection. Viruses like influenza A virus encode potent interferon antagonists, suppressing detection of viral replication such that only an incredibly small number of cells successfully produce interferons. Strikingly, even in the absence of its major interferon antagonist, NS1, influenza still only induces an interferon response in a minority of infected cells. Using a combination of single-cell RNAseq, flow cytometry, and classical bulk methods, we explored how heterogeneity, both genetic and phenotypic, in a virus lacking NS1, explains the observed rarity of the interferon response. In doing so, we find that, in the absence of NS1, viral replication is absolutely required to induce an interferon response, and that both active export and the presence of defective viral genomes can greatly enhance, but are not strictly required, for the detection of this virus. The confluence of events which we find maximizes the host response is incredibly unlikely to occur alongside the absence of the NS1 protein via the most common forms of genetic variation in the viral population, perhaps explaining, in part, how a virus with such a high error rate nevertheless has such a high rate of success at evading cell intrinsic innate immunity.

microbiology↗

bioRxiv2022

Substrate topology and cognate immunity modulate antibacterial toxicity of Tae cell wall hydrolases

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

biochemistry↗

bioRxiv2021

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Parasitic elements of the viral population which are unable to replicate on their own yet rise to high frequencies, defective interfering particles are found in a variety of different viruses. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during packaging reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.

microbiology↗

bioRxiv2018

Single-cell virus sequencing of influenza infections that trigger innate immunity

The outcome of viral infection is extremely heterogeneous, with infected cells only sometimes activating innate immunity. Here we develop a new approach to assess how the genetic variation inherent in viral populations contributes to this heterogeneity. We do this by determining both the transcriptome and full-length sequences of all viral genes in single influenza-infected cells. Most cells are infected by virions with defects such as amino-acid mutations, internal deletions, or failure to express a gene. We identify instances of each type of defect that increase the likelihood that a cell activates an innate-immune response. However, immune activation remains stochastic in cells infected by virions with these defects, and sometimes occurs even when a cell is infected by a virion that expresses unmutated copies of all genes. Our work shows that viral genetic variation substantially contributes to but does not fully explain the heterogeneity in single influenza-infected cells.

microbiology↗

bioRxiv2017

Extreme heterogeneity of influenza virus infection in single cells

Viral infection can dramatically alter a cells transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of Influenza virus infection. We 1nd extremely wide cell-to-cell variation in production of viral gene transcripts - viral transcripts compose less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, and this gene absence partially explains variation in viral transcriptional load. Despite variation in total viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells.

genomics↗