Influenza A defective viral genomes and non-infectious particles are increased by host PI3K inhibition via anti-cancer drug alpelisib.

RNA viruses produce abundant defective viral genomes during replication, setting the stage for interactions between viral genomes that alter the course of pathogenesis. Harnessing these interactions to develop antivirals has become a recent goal of intense research focus. Despite decades of research, the mechanisms that regulate the production and interactions of Influenza A defective viral genomes are still unclear. The role of the host is essentially unexplored; specifically, it remains unknown whether host metabolism can influence the formation of defective viral genomes and the particles that house them. To address this question, we manipulated host cell anabolic signaling activity and monitored the production of defective viral genomes and particles by A/H1N1 and A/H3N2 strains, using a combination of single-cell immunofluorescence quantification, third-generation long-read sequencing, and the cluster-forming assay, a method we developed to titer defective and fully-infectious particles simultaneously. Here we show that alpelisib (Piqray), a highly selective inhibitor of mammalian Class 1a phosphoinositide-3 kinase (PI3K) receptors, significantly changed the proportion of defective particles and viral genomes (specifically deletion-containing viral genomes) in a strain-specific manner, under conditions that minimize multiple cycles of replication. Alpelisib pre-treatment of cells led to an increase in defective particles in the A/H3N2 strain, while the A/H1N1 strain showed a decrease in total viral particles. In the same infections, we found that defective viral genomes of polymerase and antigenic segments increased in the A/H1N1 strain, while the total particles decreased suggesting defective interference. We also found that the average deletion size in polymerase complex viral genomes increased in both the A/H3N2 and A/H1N1 strains. The A/H1N1 strain, additionally showed a dose-dependent increase in total number of defective viral genomes. In sum, we provide evidence that host cell metabolism can increase the production of defective viral genomes and particles at an early stage of infection, shifting the makeup of the infection and potential interactions among virions. Given that Influenza A defective viral genomes can inhibit pathogenesis, our study presents a new line of investigation into metabolic states associated with less severe flu infection and the potential induction of these states with metabolic drugs.

Influenza A defective viral genome production is altered by metabolites, metabolic signaling molecules, and cyanobacteria extracts.

RNA virus infections are composed of a diverse mix of viral genomes that arise from low fidelity in replication within cells. The interactions between "defective" and full-length viral genomes have been shown to shape pathogenesis, leading to intense research into employing these to develop novel antivirals. In particular, Influenza A defective viral genomes (DVGs) have been associated with milder clinical outcomes. Yet, the full potential of DVGs as broad-spectrum antivirals remains untapped due to the unknown mechanisms of their de novo production. Much of the research into the factors affecting defective viral genome production has focused on the virus, while the role of the host has been neglected. We recently showed that altering host cell metabolism away from pro-growth pathways using alpelisib increased the production of Influenza A defective viral genomes. To uncover other drugs that could induce infections to create more DVGs, we subjected active influenza infections of the two circulating human subtypes (A/H1N1 & A/H3N2) to a screen of metabolites, metabolic signaling molecules, and cyanobacteria-derived biologics, after which we quantified the defective viral genomes (specifically deletion-containing viral genomes, DelVGs) and total viral genomes using third generation long-read sequencing. Here we show that metabolites and signaling molecules of host cell central carbon metabolism can significantly alter DelVG production early in Influenza A infection. Adenosine, emerged as a potent inducer of defective viral genomes, significantly amplifying DelVG production across both subtypes. Insulin had similar effects, albeit subtype-specific, predominantly enhancing polymerase segment DVGs in TX12 infections. Tricarboxylic Acid (TCA) cycle inhibitors 4-octyl itaconate and UK5099, along with the purine analog favipiravir, increased total viral genome production across subtypes. Cyanobacterial extracts primarily affected DVG and total viral genome production in TX12, with a specific, almost complete shutdown of influenza antigenic segments. These results underscore the influence of host metabolic pathways on DVG production and suggest new avenues for antiviral intervention, including PI3K-AKT and Ras-MAPK signaling pathways, TCA cycle metabolism, purine-pyrimidine metabolism, polymerase inhibition, and cyanotherapeutic approaches. More broadly, our findings suggest that the social interactions observed between defective and full-length viral genomes, depend not only on the viral actors, but can be altered by the stage provided by the host. Our study advances our fundamental understanding of DVG production mechanisms and highlights the potential of targeting host metabolism to develop broad-spectrum influenza therapeutics.

Influenza A virus reassortment is strain dependent.

RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange can occur when genome segments from distinct strains reassort in coinfected cells. Predicting potential genomic reassortment between influenza strains has been a long-standing goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained elusive. To address this challenge, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains encompassing two pandemics (swine and avian origin), three specific epidemics, and both circulating human subtypes A/H1N1 and A/H3N2. We found that reassortment frequency (the proportion of reassortants generated) is an emergent property of specific pairs of strains where strain identity is a predictor of reassortment frequency. We detect little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment frequencies were, on average, similar. Instead, our data suggest that certain strains bias the reassortment frequency up or down, independently of the coinfecting partner. We observe that viral productivity is also an emergent property of coinfections, but uncorrelated to reassortment frequency; thus viral productivity is a separate factor affecting the total number of reassortants produced. Assortment of individual segments among progeny and pairwise segment combinations within progeny generally favored homologous combinations. These outcomes were not related to strain similarity or shared subtype but reassortment frequency was closely correlated to the proportion of both unique genotypes and of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential to realize the goal of predicting influenza virus evolution resulting from segment exchange.

5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium.

5-Aminosalicylic acid (5-ASA), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, is a widely used first-line medication for the treatment of ulcerative colitis, but its anti-inflammatory mechanism is not fully resolved. Here, we show that 5-ASA ameliorates colitis in dextran sulfate sodium (DSS)-treated mice by activating PPAR-γ signaling in the intestinal epithelium. DSS-induced colitis was associated with a loss of epithelial hypoxia and a respiration-dependent luminal expansion of Escherichia coli, which could be ameliorated by treatment with 5-ASA. However, 5-ASA was no longer able to reduce inflammation, restore epithelial hypoxia, or blunt an expansion of E. coli in DSS-treated mice that lacked Pparg expression specifically in the intestinal epithelium. These data suggest that the anti-inflammatory activity of 5-ASA requires activation of epithelial PPAR-γ signaling, thus pointing to the intestinal epithelium as a potential target for therapeutic intervention in ulcerative colitis.IMPORTANCE An expansion of Enterobacterales in the fecal microbiota is a microbial signature of dysbiosis that is linked to many noncommunicable diseases, including ulcerative colitis. Here, we used Escherichia coli, a representative of the Enterobacterales, to show that its dysbiotic expansion during colitis can be remediated by modulating host epithelial metabolism. Dextran sulfate sodium (DSS)-induced colitis reduced mitochondrial activity in the colonic epithelium, thereby increasing the amount of oxygen available to fuel an E. coli expansion through aerobic respiration. Activation of epithelial peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling with 5-aminosalicylic acid (5-ASA) was sufficient to restore mitochondrial activity and blunt a dysbiotic E. coli expansion. These data identify the host's epithelial metabolism as a potential treatment target to remediate microbial signatures of dysbiosis, such as a dysbiotic E. coli expansion in the fecal microbiota.