Abstract
To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.
Highlights
The threat of influenza infection is felt globally, and the disease leads to an estimated 3–5 million cases of severe illness and about 250,000 –500,000 deaths each year [1]
Influenza A virus (IAV) infection resulted in host cell cycle arrest in G0/G1 phase, which was accompanied by cyclin D3 relocalization and degradation
It is of considerable interest to identify intracellular interactors of matrix protein 2 (M2)-cytoplasmic tail (CT), to elucidate cellular components that are exploited by influenza, and to understand host defenses involved in restricting the virus life cycle
Summary
Y2H, yeast two-hybrid; TPCK, L-1-tosylamide-2phenylethyl chloromethyl ketone; CT, cytoplasmic tail; CDK, cyclin-dependent kinase; IAV, influenza A virus; MOI, multiplicity of infection; p.i., postinfection; Rb, retinoblastoma; ppRb, hyperphosphorylated Rb; pRb, hypophosphorylated Rb; CKI, cyclin-dependent kinase inhibitor; NT, nontargeting; HBEC, human bronchial epithelial cell; ISG, interferon-stimulated gene; contig, group of overlapping clones; MDCK, Madin-Darby canine kidney; pAb, polyclonal antibody; FAM, 6-carboxyfluorescein; TAMRA, tetramethylrhodamine; ANOVA, analysis of variance. We reasoned that the integral membrane proteins of the viral envelope would interact with cellular factors at various stages: endosomal fusion and release of the genetic material during entry, transport from endoplasmic reticulum to the plasma membrane, and assembly and budding of nascent virions. Rossman et al [28] reported a role of M2-CT in mediating cholesterol-dependent alteration in membrane curvature at the neck of budding virions, leading to host ESCRT pathway-independent membrane scission. These studies provide evidence that influenza M2, especially the CT domain, plays a critical role in multiple steps of the virus life cycle. Our results suggest a novel function of cyclin D3 that is beyond its classical function in cell cycle regulation
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