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

Abstract

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.

Figure 1.. Control infections were highly repeatable.

A. Production of influenza defective viral genomes (here measured as proportion of Deletion-containing Viral Genomes (DelVGs) in strain CA09 (H1N1pdm) after 18 hr infection (no trypsin) mocktreated with water vehicle (left panel) and DMSO vehicle (right panel). B. Production of influenza Total Viral Genomes (TVG) in strain CA09 (H1N1pdm) after 18 hr infection (no trypsin) mock-treated with water vehicle (left panel) and DMSO vehicle (right panel). Point colors represent data from three independent infections conducted on different days (i.e. bioreplicates).

Figure 2.. Drug treatment affects defective viral genomes and total viral genomes.

A. Production of influenza defective viral genomes (here measured as proportion of Deletion-containing Viral Genomes (DelVGs) after 18 hr infection; each point is the proportion of DelVGs in each influenza segment (all segments included in each treatment). B. Production of influenza Total Viral Genomes (TVG) after 18 hr infection; each point is the total number of viral genomes of each influenza segment (all segments included in each treatment). The first two treatments (left to right) are vehicle control infections. Point colors represent data from three independent infections conducted on different days (i.e. bioreplicates). The shape of the points represents the strain (circles = CA09(H1N1pdm), diamonds = TX12(H3N2)). The position of points is randomly jittered horizontally for better visibility.

Figure 3.. Adenosine and insulin increase polymerase complex defective viral genome segments.

Production of influenza defective viral genomes (here measured as proportion of Deletion-containing Viral Genomes (DelVGs) after 18 hr infection; each point is the proportion of DelVGs in each influenza segment. Point colors represent data from three independent infections conducted on different days (i.e. bioreplicates).

Figure 4.. TCA cycle flux inhibitors 4-OI and UK5099 & purine analog favipiravir increase total viral genomes in A/H1N1 and A/H3N2 strains.

Production of influenza Total Viral Genomes (TVG) after 18 hr infection; each point is the count of total viral genomes (all segments included in each treatment). Point colors represent data from three independent infections conducted on different days (i.e. bioreplicates).

Figure 5.. Cyanobacterial extracts suppress total viral genome production in A/H3N2 strain antigenic segments.

Production of influenza Total Viral Genomes (TVG) after 18 hr infection; each point is the count of total viral genomes in each influenza segment. Point colors represent data from three independent infections conducted on different days (i.e. bioreplicates).