Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection

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.

Beyond Channel Activity: Protein-Protein Interactions Involving Viroporins

“…However, the role of BST-2 in limiting IAV release has been disputed (Watanabe et al 2011), perhaps due to IAV strain-specific susceptibility to BST-2 restriction. This interaction was confirmed by orthogonal assays, and using chimeric and truncated M2, the regions Fan et al (2017)] involved in BST-2 downregulation were proposed to be within the M2 extracellular and TMDs.…”

“…Results from another yeast two-hybrid screen using the M2 cytoplasmic tail as a bait have identified the human cell cycle regulator cyclin D3 as a binder, and their interaction has been confirmed in infected cells by immunoprecipitation assays (Fan et al 2017). Using siRNA-mediated knockdown of cyclin D3 expression, the study proposed that cyclin D3 is a negative modulator of IAV infection, competing with M1 for binding to M2 in transfected cells and therefore disrupting the important M1-M2 interaction in the context of an IAV infection.…”

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle

“…It has been reported that SLD5 interacts with Psf1, Psf2, and Psf3 (Choi et al, 2007;Gouge & Christensen, 2010;Joshi et al, 2016;Takayama et al, 2003). Previous studies have shown that IAV infection induces host cell G0/G1 phase arrest (Fan et al, 2017;He et al, 2010;Jiang et al, 2013). Some bacterial virus can shut off host DNA replication to arrest host growth (Belley et al, 2006;Seco et al, 2013;Yano & Rothman-Denes, 2011).…”

“…Although the strength of the cell cycle arrest varies among different influenza virus strains, it is possible that more than one viral protein is involved in this process in certain strains (Fan et al, 2017;He et al, 2010;Jiang et al, 2013;Wang et al, 2012). The NS1 protein from WSN virus has been shown to arrest host cell cycle mainly through interfering with the RhoA/pRb signalling cascade (Jiang et al, 2013).…”

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

“…This approach reduces the likelihood of resistance since host genes are unlikely to mutate as fast as viral ones. Many scientists have used the yeast two-hybrid system to identify host interactors of individual viral proteins: NS1 [144][145][146][147][148][149][150][151][152][153][154][155][156][157][158], NS2 [129,144,[159][160][161], NP [162][163][164][165][166][167][168], PA [169][170][171][172], PB1 [173], PB2 [174], NA [175], M1 [176][177][178][179], M2 [180][181][182][183][184] and PB1-F2 [185][186][187][188][189]. Several studies applied the two-hybrid syste...…”