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

Wohlgemuth, Nicholas; Lane, Andrew P.; Pekosz, Andrew

doi:10.1128/jvi.01425-18

Cited by 24 publications

(21 citation statements)

References 79 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Assuming that the amount of M2 at the plasma membrane is a limiting factor for the production of infectious virus particles, the reduced surface expression of M2 ALPS and M2 Epsin might partly account for the reduced virus titers. This is consistent with a recent report describing that altering the expression and the intracellular targeting of M2 has major effects on virus replication (64).…”

Section: Discussionsupporting

confidence: 93%

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Siche

Møller

et al. 2019

Preprint

View full text Add to dashboard Cite

M2 of influenza virus functions as proton channel during virus entry. In addition, an 1 amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the 2 assembly site where it inserts into the inner membrane leaflet to induce curvature that causes 3 virus scission. Since vesicularisation of membranes can be performed by a variety of 4 amphiphilic peptides we used reverse genetics to investigate whether they can substitute for 5 M2´s helix. 6 Virus could not be generated if M2´s helix was deleted or replaced by a peptide predicted not 7 to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was 8 exchanged by helices known to induce membrane curvature. Infectious virus titers were 9 marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor, from the 10 Epsin N-Terminal Homology domain or the non-natural membrane inducer RW16. 11Transmission EM of infected cells did not reveal unequivocal evidence that virus budding or 12 membrane scission was disturbed in any of the mutants. Instead, individual virus mutants 13 exhibit other defects in M2, such as reduced surface expression, incorporation into virus 14 particles and ion channel activity. The protein composition and specific infectivity was also 15 altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is 16 essential for virus replication, but other helices can replace its basic (curvature-inducing) 17 function. 18 3 Importance 19Influenza is unique among enveloped viruses since it does not rely on the cellular ESCRT-20 machinery for budding. Instead viruses encode their own scission machine, the M2 protein. 21M2 is targeted to the edge of the viral assembly site where it inserts an amphiphilic helix into 22 the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission 23 of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins 24 with only small effects on virus replication. No evidence was obtained that budding is 25 disturbed, but individual mutants exhibit other defects in M2 which explain the reduced virus 26 titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by 27 irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic 28 helix to induce membrane curvature, but its biophysical properties are more important than 29 the amino acid sequence. 30 4 71membrane curvature as shown by preferential binding of M2 to small unilamellar vesicles 72 (SUVs) having a small diameter and by molecular dynamics simulations (31, 32). More 73 specifically, clusters of M2 molecules are excluded from regions with negative curvature, i. e. 74 the outward part of a budding virus particle, but rather accumulate at membrane regions with 75 positive curvature, i. e. the neck of a budding virus (33). 76Positioning of M2 at the edge of the viral budding zone could then entail the scission of virus 77 particles: the amphiphilic helix...

show abstract

Section: Discussionsupporting

confidence: 93%

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Siche

Møller

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The polarized targeting of viral proteins is critical for the pathogenesis of several viruses. The influenza A virus M2 protein must be at the apical side of the host cell for the optimal production of infectious particles (46). Similarly, measles virus is released from the apical membranes of polarized epithelial cells by hijacking Rab11A-positive recycling endosomes (47).…”

Section: Discussionmentioning

confidence: 99%

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

Capelli

Marion

Dubois

et al. 2019

J Virol

View full text Add to dashboard Cite

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host. IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients’ blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

show abstract

“…cDNA was generated from the pHH21-NA flag plasmids with Q5 Hot-Start PCR (NEB). This cDNA product was then cloned into the mammalian expression vector pCAGGS for transient transfection experiments as previously described [51].…”

Section: Methodsmentioning

confidence: 99%

“…For generation of recombinant virus seed stocks, 400ul of plaque picked virus was added to confluent MDCK cells plated in 6 well plates and infected for 1hr as previously described [49, 51]. The plaque pick inoculum was removed and infection media (IM) was added.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Neuraminidase antigenic drift of Influenza A virus H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding

Powell

Pekosz

2020

Preprint

Self Cite

View full text Add to dashboard Cite

27 In the 2014-2015 influenza season a novel neuraminidase (NA) genotype emerged in 28 the Johns Hopkins Center of Excellence for Influenza Research and Surveillance (JH 29 CEIRS) surveillance network as well as globally. This novel genotype encoded a 30 glycosylation site at position 245-247 in the NA protein from clade 3c.2a H3N2 viruses.31 In the years following the 2014-2015 season, this novel NA glycosylation genotype 32 quickly dominated the human H3N2 population of viruses. To assess the effect this 33 novel glycosylation has on virus fitness and antibody binding, recombinant viruses with 34 (NA Gly+) or without (NA Gly-) the novel NA glycosylation were created. Viruses with 35 the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, 36 differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA 37 Gly-genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked 38 human and rabbit monoclonal antibodies that target the enzymatic site from binding to 39 their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly 40 lower enzymatic activity compared to viruses with the 245 NA Gly-genotype. Human 41 monoclonal antibodies that target residues near the 245 NA glycosylation were less 42 effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an 43 NA Gly+ protein compared to ones encoding NA Gly-protein. Additionally, a 44 recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic 45 inhibition from convalescent serum from H3N2-infected humans compared to viruses 46 with the 245 NA Gly-genotype. Finally, the 245 NA Gly+ protected from NA antibody 47 mediated virus neutralization. These results suggest that while the 245 NA Gly+ 48 decreases virus replication in hNECs and decreases enzymatic activity, the 49 glycosylation blocks the binding of monoclonal and human serum NA specific antibodies 50 that would otherwise inhibit enzymatic activity and virus replication. Influenza virus infects millions of people worldwide and leads to thousands of deaths 74 and millions in economic loss each year. During the 2014/2015 season circulating 75 human H3N2 viruses acquired a novel mutation in the neuraminidase (NA) protein. This 76 mutation has since fixed in human H3N2 viruses. This mutation at position 245 through 77 247 in the amino acid sequence of NA encoded an N-linked glycosylation. Here, we 78 studied how this N-linked glycosylation impacts virus fitness and protein function. We 79 found that this N-linked glycosylation on the NA protein decreased viral replication 80 fitness on human nasal epithelial cells (hNEC) but not immortalized Madin-Darby 81 Canine Kidney (MDCK) cells. We also determined this glycosylation decreases NA 82 enzymatic activity, enzyme kinetics and affinity for substrate. Furthermore, we show that 83 this N-linked glycosylation at position 245 blocks some NA specific inhibitory antibodies 84 from binding to the protein, inhibiting enzyma...

show abstract

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

Cited by 24 publications

References 79 publications

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

Neuraminidase antigenic drift of Influenza A virus H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding

Contact Info

Product

Resources

About