Unpredictable
influenza pandemics, annual epidemics, and sporadic poultry-to-human
avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new
antiviral approaches. Targeting cellular pathways and processes is a promising
antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative
antiviral mode of action of the drug(s).
Ribonucleases (RNases) of different origins not only demonstrate
antiviral effects that are mediated by the direct
RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the
RNase-affected cell
proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the
antiviral effect employed by the small extra-cellular
ribonuclease of Bacillus pumilus (
binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon
binase treatment were performed. We compared
proteomes of mock-treated,
binase-treated, virus-infected, and virus-infected
binase-treated cells to determine the
proteins affected by IAV and/or
binase. In general, IAV demonstrated a downregulating strategy towards cellular
proteins, while
binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular
protein sets were defined and assigned to their biological function; a possible interconnection with the progression of
viral infection was conferred. Most of the
proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated
proteins upon the
binase treatment (e.g., AKR1B10,
CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The
antiviral activity of
binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear
ribonucleoprotein export (NCL, NPM1, Nup205, and Bax
proteins involved) and cytoskeleton remodeling (
RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle
infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that
binase exerts its
antiviral effects in multiple ways.