Abstract
BackgroundInfluenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.MethodsIn this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes.ResultsSeventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).ConclusionsTen human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.
Highlights
Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species
The two NP open reading frame (ORF) were amplified by PCR, and the resulting PCR products were separately inserted by recombination cloning into Yeast two-hybrid (Y2H) bait vector pGBKT7 as a C-terminal fusion with Gal4p’s DNA binding domain
This Y2H screen identified multiple candidate host proteins that were previously identified in genome-wide screens as playing a role in the IAV infection cycle (PAICS, ATP1B1, SLC30A9, and TRA2B), while identifying new potential interactors with IAV nucleoprotein (ACOT13, CKAP5, KCTD9, PA28B, MSANDTD3, and FLJ30306)
Summary
Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies. In addition to seasonal influenza A viruses, pandemic strains periodically appear causing increased mortality rates. Due to influenza A’s potential for mortality, high mutation rates (resulting in genetic drift) and pandemic potential (resulting from genetic reassortment), it is critical to learn more about the virus, especially as it pertains to virulence, Generous et al Virology Journal (2014) 11:228 transmissibility, and identification of potential targets for development of therapeutics
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