scholarly journals Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Pengyang Zhu ◽  
Libin Liang ◽  
Xinyuan Shao ◽  
Weiyu Luo ◽  
Shuitao Jiang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Influenza A Virus ◽  
Influenza A ◽  
Secretory Pathway ◽  
Host Factors ◽  
Apical Plasma Membrane ◽  
M2 Protein ◽  
Internal Deletion

ABSTRACT Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice. IMPORTANCE The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

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

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Nicholas Wohlgemuth ◽  
Andrew P. Lane ◽  
Andrew Pekosz
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Apical Plasma Membrane ◽  
M2 Protein ◽  
Particle Release

ABSTRACTThe 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.IMPORTANCEInfluenza 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.

scholarly journals MARCH8 inhibits influenza A virus infection by targeting viral M2 protein for ubiquitination-dependent degradation in lysosomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoman Liu ◽  
Fengwen Xu ◽  
Lili Ren ◽  
Fei Zhao ◽  
Yu Huang ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Cellular Membrane ◽  
Immune Defense ◽  
M2 Protein ◽  
E3 Ligases ◽  
Underlying Mechanisms ◽  
The Stability

AbstractThe membrane-associated RING-CH (MARCH) proteins are E3 ligases that regulate the stability of various cellular membrane proteins. MARCH8 has been reported to inhibit the infection of HIV-1 and a few other viruses, thus plays an important role in host antiviral defense. However, the antiviral spectrum and the underlying mechanisms of MARCH8 are incompletely defined. Here, we demonstrate that MARCH8 profoundly inhibits influenza A virus (IAV) replication both in vitro and in mice. Mechanistically, MARCH8 suppresses IAV release through redirecting viral M2 protein from the plasma membrane to lysosomes for degradation. Specifically, MARCH8 catalyzes the K63-linked polyubiquitination of M2 at lysine residue 78 (K78). A recombinant A/Puerto Rico/8/34 virus carrying the K78R M2 protein shows greater replication and more severe pathogenicity in cells and mice. More importantly, we found that the M2 protein of the H1N1 IAV has evolved to acquire non-lysine amino acids at positions 78/79 to resist MARCH8-mediated ubiquitination and degradation. Together, our data support the important role of MARCH8 in host anti-IAV intrinsic immune defense by targeting M2, and suggest the inhibitory pressure of MARCH8 on H1N1 IAV transmission in the human population.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2903
Author(s):  
Maggie C. Duncan ◽  
Pascal Amoa Onguéné ◽  
Ibuki Kihara ◽  
Derrick N. Nebangwa ◽  
Maya E. Naidu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Drug Resistant ◽  
M2 Protein ◽  
Virtual Screen ◽  
Chebulagic Acid ◽  
Virus Strains

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

scholarly journals The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

2010 ◽  
Vol 84 (12) ◽  
pp. 5848-5859 ◽  
Author(s):  
Emily A. Bruce ◽  
Paul Digard ◽  
Amanda D. Stuart
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Influenza A Virus ◽  
Membrane Trafficking ◽  
Influenza A ◽  
Spherical Particles ◽  
Actin Dynamics ◽  
Apical Plasma Membrane ◽  
Thin Sections ◽  
Virus Particles

ABSTRACT Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.

scholarly journals Role of neuromedin B and its receptor in the innate immune responses against influenza A virus infection in vitro and in vivo

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Guihong Yang ◽  
Huipeng Huang ◽  
Mengyao Tang ◽  
Zifeng Cai ◽  
Cuiqin Huang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
A549 Cells ◽  
Virus Challenge ◽  
Neuromedin B ◽  
Influenza A Virus Infection

Abstract The peptide neuromedin B (NMB) and its receptor (NMBR) represent a system (NMB/NMBR) of neuromodulation. Here, it was demonstrated that the expression of NMBR in cells or murine lung tissues was clearly upregulated in response to H1N1/PR8 influenza A virus infection. Furthermore, the in vitro and in vivo activities of NMB/NMBR during PR8 infection were investigated. It was observed that A549 cells lacking endogenous NMBR were more susceptible to virus infection than control cells, as evidenced by the increased virus production in the cells. Interestingly, a significant decrease in IFN-α and increased IL-6 expression were observed in these cells. The role of this system in innate immunity against PR8 infection was probed by treating mice with NMB. The NMB-treated mice were less susceptible to virus challenge, as evidenced by increased survival, increased body weight, and decreased viral NP expression compared with the control animals. Additionally, the results showed that exogenous NMB not only enhanced IFN-α expression but also appeared to inhibit the expression of NP and IL-6 in PR8-infected cells and animals. As expected, opposing effects were observed in the NMBR antagonist-treated cells and mice, which further confirmed the effects of NMB. Together, these data suggest that NMB/NMBR may be an important component of the host defence against influenza A virus infection. Thus, these proteins may serve as promising candidates for the development of novel antiviral drugs.

scholarly journals Semi-continuous propagation of influenza A virus and its defective interfering particles: analyzing the dynamic competition to select candidates for antiviral therapy

2021 ◽  
Author(s):  
Lars Pelz ◽  
Daniel Rüdiger ◽  
Tanya Dogra ◽  
Fadi G. Alnaji ◽  
Yvonne Genzel ◽  
...  
Keyword(s):  
Antiviral Therapy ◽  
Influenza A Virus ◽  
Influenza A ◽  
De Novo ◽  
Competitive Inhibition ◽  
Antiviral Treatment ◽  
Small Scale ◽  
Internal Deletion ◽  
Defective Interfering Particles

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that comprise an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon co-infection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in a STV co-infection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage. Yet, an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy than DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. Importance Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against non-homologous viruses, like SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro , and we suggest them for effective antiviral therapy.

The role of the N-terminal caspase cleavage site in the nucleoprotein of influenza A virus in vitro and in vivo

2007 ◽  
Vol 153 (3) ◽  
pp. 427-434 ◽  
Author(s):  
A. S. Lipatov ◽  
H.-L. Yen ◽  
R. Salomon ◽  
H. Ozaki ◽  
E. Hoffmann ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Cleavage Site ◽  
Influenza A ◽  
Caspase Cleavage ◽  
Caspase Cleavage Site

scholarly journals Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
John T. Wren ◽  
Lance K. Blevins ◽  
Bing Pang ◽  
Ankita Basu Roy ◽  
Melissa B. Oliver ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Middle Ear ◽  
Influenza A ◽  
Nasal Colonization ◽  
Ear Infection ◽  
Content Type ◽  
Public Health Burden ◽  
Middle Ear Infection

ABSTRACT Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens.

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