Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail

Liu, Hsuan; Grantham, Michael L.; Pekosz, Andrew

doi:10.1128/jvi.00858-17

Cited by 26 publications

(28 citation statements)

References 47 publications

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“…There are other mutations in M2 apart from residue 78 in recent H1N1 isolates. A recent study indicated that a lethal mutation in the M2 cytoplasmic tail can be compensated for by mutations in the M1 protein to restore the production of infectious virus (34). Together, the K78 mutation plus other mutations may affect virus replication.…”

Section: Discussionmentioning

confidence: 99%

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

Huang

et al. 2018

J Virol

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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.

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Section: Discussionmentioning

confidence: 99%

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

Huang

et al. 2018

J Virol

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“…RNA from virus particles secreted into the supernatant was isolated and reverse transcribed into cDNA as described previously (78). Virus supernatant from high-MOI infections was concentrated through a 35% sucrose cushion at 118,000 ϫ g for 1 h in an Optima Max-TL Beckman Ultracentrifuge.…”

Section: Methodsmentioning

confidence: 99%

“…Virus supernatant from high-MOI infections was concentrated through a 35% sucrose cushion at 118,000 ϫ g for 1 h in an Optima Max-TL Beckman Ultracentrifuge. The qPCR assay was performed as described previously (78), except the sample volumes and primers described above were used. All samples were mixed with 4ϫ SDS-PAGE sample buffer containing dithiothreitol (DTT; ThermoFisher) before heating for 5 min at 100°C.…”

Section: Methodsmentioning

confidence: 99%

Influenza A Virus M2 Protein Apical Targeting Is Required for Efficient Virus Replication

Wohlgemuth

Lane

Pekosz

2018

J Virol

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The influenza A virus (IAV) M2 protein is a multifunctional protein with critical roles in virion entry, assembly, and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to those of wild-type virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type-independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells. Influenza A virus assembly and particle release occur at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus, HA, NA, and M2, are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release, while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.

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“…In contrast, MDM showed very few buds on their surface (Figure 4), suggesting that in MDM, an earlier role for M2 in virus particle assembly is more prominent than in epithelial cells. Consistent with this early role of M2 in IAV assembly, M2 functions in recruitment of M1 and vRNP to assembly sites (41, 58–63), which is important for initiation of IAV particle assembly or elongation of filamentous particles (58, 60, 65–71). A defect in incorporation of M1 and/or vRNP into budding virus particles due to the failure of M2 recruitment may also explain the reduction in infectivity per particle observed for MDM-derived virus relative to dTHP1-derived virus (Figure 2B).…”

Section: Discussionmentioning

confidence: 64%

A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

Bedi

Noda

Kawaoka

et al. 2017

Preprint

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18The primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In 19 contrast to epithelial cells, productive virus infection of most IAV strains is either 20 completely blocked or highly inefficient in macrophages. The exact nature of the defect 21 in IAV replication that leads to inefficient production of progeny virus particles in human 22 macrophages remains to be determined. In this study, we showed that primary human 42show that primary human MDM are not permissive to IAV replication due to a defect at 43 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/213447 doi: bioRxiv preprint first posted online Nov. 2, 2017; the virus particle formation step. This defect is specific to primary human macrophages, 44 since a human monocyte-derived cell line differentiated into macrophage-like 45 morphology supports IAV particle formation. Further, comparison of these cell types 46suggests that the defect in virus particle assembly in MDM is at least partly due to 47 inefficient association between two viral glycoproteins, HA and M2, on the surface of 48 these cells. Overall, by identifying the viral protein interaction susceptible to cell-type-49 specific differences, our study suggests the possibility that the interactions between viral 50 components can be targeted to block IAV assembly in infected cells. 52 53peer-reviewed)

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Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail

Cited by 26 publications

References 47 publications

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

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

Influenza A Virus M2 Protein Apical Targeting Is Required for Efficient Virus Replication

A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

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