Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein Is Crucial for Production of Infectious Virus Particles

Abstract

Virus replication is mediated by interactions between the virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in the production of infectious virus particles. M2-K78R mutant progeny contained more hemagglutinin (HA) proteins, less viral RNAs, and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination by electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with the M1 protein to a lesser extent than for the wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal levels of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that the M2-K78R mutant virus induced autophagy and apoptosis earlier than did the wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating the efficient packaging of the viral genome into virus particles and the timing of virus-induced cell death. Annual epidemics and recurring pandemics of influenza viruses represent very high global health and economic burdens. The influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in virus entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N terminus of M2 proteins. However, viruses that are resistant to rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

To TRIM or not to TRIM: the balance of host–virus interactions mediated by the ubiquitin system

“…Additional viral components outside of the replication complex have shown a requirement for ubiquitination that can directly impact viral propagation. Identification of K78 on the cytoplasmic domain of the IAV M2 protein as a target for ubiquitination uncovered a role for ubiquitin in the successful formation of progeny virions [195]. Mutant viruses harbouring a K78R switch in their M2 protein produced severely flawed viral particles that lacked vRNA and vRNPs and otherwise appeared empty under electron microscopy observation [195].…”

Pleiotropic roles of the ubiquitin-proteasome system during viral propagation

“…To date, many viral proteins have been reported to be ubiquitinated, including influenza A virus-encoded NP, M2 and PB1 [25][26][27][28], Dengue Virus-encoded NP [29], and 54 viral proteins encoded by the wellcharacterised cowpox virus strain [30]. In ubiquitinated proteins, Ub is frequently found to be covalently conjugated to Lys residues, but non-Lys residues of substrates such as serine (Ser), threonine (Thr) and cysteine (Cys) are also modified in rare cases.…”

“…For example, BPLF1 of three DUBs encoded by EBV genome was reported to contributes to innate immune evasion through interference with toll-like receptor signalling [56], and arterivirus papain-like protease 2 with the activity of DUB can suppresses the innate immune response in infected host cells [57]. Moreover, NP, M2 and PB1 proteins of influenza A virus are ubiquitinated, and this affects replication of the viral genome [25][26][27][28]. Furthermore, Kirui et al revealed that ubiquitination can up-regulate influenza virus polymerase activity, directly enhancing viral RNA synthesis [58].…”

Acetylation at K108 of the NS1 protein is important for the replication and virulence of influenza virus

“…Post-translational modification is important for protein function, stability, cellular localization, and protein-protein interactions. Recent studies have shown that posttranslational modifications of viral proteins modulate the virus life cycle, e.g., phosphorylation of influenza viral proteins (NS1, M1, and NP) plays important roles in virus replication [35][36][37][38]. The ubiquitination of NP and M2 proteins is crucial for viral RNA replication and the production of infectious virus particles [37].…”