Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses

ABSTRACT The influenza virus neuraminidase (NA) is a tetrameric, virus surface glycoprotein possessing receptor-destroying activity. This enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has resulted in temperature-sensitive (ts) proteins, we asked whether mutation of cysteine residues in the influenza virus NA would yield ts mutants. The ability to rationally design tight and stable ts mutations could facilitate the creation of efficient helper viruses for influenza virus reverse genetics experiments. We generated a series of cysteine-to-glycine mutants in the influenza A/WSN/33 virus NA. These were assayed for neuraminidase activity in a transient expression system, and active mutants were rescued into infectious virus by using established reverse genetics techniques. Mutation of two cysteines not involved in intrasubunit disulfide bonds, C49 and C146, had modest effects on enzymatic activity and on viral replication. Mutation of two cysteines, C303 and C320, which participate in a single disulfide bond located in the β5L0,1 loop, produced ts enzymes. Additionally, the C303G and C320G transfectant viruses were found to be attenuated and ts. Because both the C303G and C320G viruses exhibited stable ts phenotypes, they were tested as helper viruses in reverse genetics experiments. Efficiently rescued were an N1 neuraminidase from an avian H5N1 virus, an N2 neuraminidase from a human H3N2 virus, and an N7 neuraminidase from an H7N7 equine virus. Thus, these cysteine-to-glycine NA mutants allow the rescue of a variety of wild-type and mutant NAs into influenza virus.

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Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

Scientific Reports ◽

10.1038/s41598-019-51877-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Mariana Baz ◽

Zeineb M’hamdi ◽

Julie Carbonneau ◽

Sophie Lavigne ◽

Christian Couture ◽

...

Keyword(s):

Animal Model ◽

Influenza Virus ◽

Reverse Genetics ◽

Polymerase Activity ◽

Influenza Viruses ◽

H3n2 Virus ◽

Wild Type Virus ◽

Virus Research ◽

H3n2 Influenza

Abstract The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

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A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF

Journal of Virology ◽

10.1128/jvi.00514-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6419-6426 ◽

Cited By ~ 32

Author(s):

Qinshan Gao ◽

Edward W. A. Brydon ◽

Peter Palese

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Chimeric Protein ◽

Influenza Viruses ◽

Viral Rna ◽

Surface Glycoprotein ◽

Influenza C Virus ◽

Major Surface

ABSTRACT Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.

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Cell Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Ferret Airborne Transmission

10.20944/preprints202103.0297.v1 ◽

2021 ◽

Author(s):

Valerie Le Sage ◽

Karen Kormuth ◽

Eric Ntruibi ◽

Juhye Lee ◽

Sheila A Frizzell ◽

...

Keyword(s):

Cell Culture ◽

Influenza Virus ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Surface Glycoprotein ◽

H3n2 Virus ◽

Airborne Transmission ◽

Acid Stability ◽

Public Health Burden ◽

Culture Adaptation

Airborne transmission of seasonal and pandemic influenza viruses is responsible for their epidemiological success and public health burden in humans. Efficient airborne transmission of H1N1 influenza virus relies on receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examine the role of HA pH of fusion on transmissibility of a cell culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decrease the acid stability of the virus from a pH of 5.5 (WT) to 5.8 (mutant). In addition, we observed that this mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, we demonstrate that the NA segment noncoding regions can impact the pH of fusion of reassortant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

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Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model

Viruses ◽

10.3390/v13050719 ◽

2021 ◽

Vol 13 (5) ◽

pp. 719

Author(s):

Valerie Le Sage ◽

Karen A. Kormuth ◽

Eric Nturibi ◽

Juhye M. Lee ◽

Sheila A. Frizzell ◽

...

Keyword(s):

Cell Culture ◽

Influenza Virus ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Surface Glycoprotein ◽

H3n2 Virus ◽

Airborne Transmission ◽

Acid Stability ◽

Public Health Burden ◽

Culture Adaptation

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

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A Live-Attenuated Prime, Inactivated Boost Vaccination Strategy with Chimeric Hemagglutinin-Based Universal Influenza Virus Vaccines Provides Protection in Ferrets: A Confirmatory Study

Vaccines ◽

10.3390/vaccines6030047 ◽

2018 ◽

Vol 6 (3) ◽

pp. 47 ◽

Cited By ~ 15

Author(s):

Raffael Nachbagauer ◽

Florian Krammer ◽

Randy Albrecht

Keyword(s):

Influenza Virus ◽

Respiratory Tract ◽

Virus Vaccine ◽

Influenza Viruses ◽

Inactivated Vaccine ◽

Upper Respiratory Tract ◽

Live Attenuated Vaccine ◽

Virus Surface ◽

Virus Challenge ◽

Attenuated Vaccine

Influenza viruses cause severe diseases and mortality in humans on an annual basis. The current influenza virus vaccines can confer protection when they are well-matched with the circulating strains. However, due to constant changes of the virus surface glycoproteins, the vaccine efficacy can drop substantially in some seasons. In addition, the current seasonal influenza virus vaccines do not protect from avian influenza viruses of human pandemic potential. Novel influenza virus vaccines that aim to elicit antibodies against conserved epitopes like the hemagglutinin stalk could not only reduce the burden of drifted seasonal viruses but potentially also protect humans from infection with zoonotic and emerging pandemic influenza viruses. In this paper, we generated influenza virus vaccine constructs that express chimeric hemagglutinins consisting of exotic, avian head domains and a consistent stalk domain of a seasonal virus. Using such viruses in a sequential immunization regimen can redirect the immune response towards conserved epitopes. In this study, male ferrets received a live-attenuated vaccine virus based on the A/Ann Arbor/6/60 strain expressing a chimeric H8/1 (cH8/1) hemagglutinin, which was followed by a heterologous booster vaccination with a cH5/1N1 formalin inactivated non-adjuvanted whole virus. This group was compared to a second group that received a cH8/1N1 inactivated vaccine followed by a cH5/1N1 inactivated vaccine. Both groups showed a reduction in viral titers in the upper respiratory tract after the A(H1N1)pdm09 virus challenge. Animals that received the live-attenuated vaccine had low or undetectable titers in the lower respiratory tract. The results support the further development of chimeric hemagglutinin-based vaccination strategies. The outcome of this study confirms and corroborates findings from female ferrets primed with a A/Leningrad/134/17/57-based live attenuated cH8/1N1 vaccine followed by vaccination with an AS03-adjuvanted cH5/1N1 split virus vaccine 10.

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Prediction, dynamics, and visualization of antigenic phenotypes of seasonal influenza viruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525578113 ◽

2016 ◽

Vol 113 (12) ◽

pp. E1701-E1709 ◽

Cited By ~ 101

Author(s):

Richard A. Neher ◽

Trevor Bedford ◽

Rodney S. Daniels ◽

Colin A. Russell ◽

Boris I. Shraiman

Keyword(s):

Influenza Virus ◽

Seasonal Influenza ◽

Sequence Data ◽

Influenza Viruses ◽

Surface Glycoprotein ◽

Amino Acid Substitutions ◽

Model Output ◽

Virus Population ◽

Web Browser ◽

Antigenic Properties

Human seasonal influenza viruses evolve rapidly, enabling the virus population to evade immunity and reinfect previously infected individuals. Antigenic properties are largely determined by the surface glycoprotein hemagglutinin (HA), and amino acid substitutions at exposed epitope sites in HA mediate loss of recognition by antibodies. Here, we show that antigenic differences measured through serological assay data are well described by a sum of antigenic changes along the path connecting viruses in a phylogenetic tree. This mapping onto the tree allows prediction of antigenicity from HA sequence data alone. The mapping can further be used to make predictions about the makeup of the future A(H3N2) seasonal influenza virus population, and we compare predictions between models with serological and sequence data. To make timely model output readily available, we developed a web browser-based application that visualizes antigenic data on a continuously updated phylogeny.

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Immunogenicity and protective activity of recombinant influenza viruses expressing fragments of ScaAB lipoprotein of group B streptococci in a mouse model

Medical academic journal ◽

10.17816/maj50307 ◽

2020 ◽

Vol 20 (3) ◽

pp. 33-42

Author(s):

E. A. Stepanova ◽

I. N. Isakova-Sivak ◽

V. A. Matyushenko ◽

A. S. Matushkina ◽

T. A. Smolonogina ◽

...

Keyword(s):

Amino Acids ◽

Bacterial Infections ◽

Reverse Genetics ◽

Mdck Cells ◽

Influenza Viruses ◽

Effective Vaccine ◽

Temperature Sensitive ◽

Protective Activity ◽

Group B Streptococci ◽

Group B

Group B streptococci (GBS) cause a number of serious diseases in humans. The development of an effective vaccine against GBS requires special approaches. In the present study, three recombinant influenza viruses were constructed on the backbone of H7N9 live attenuated influenza vaccine (LAIV) strain expressing fragments of the ScaAB lipoprotein of Streptococcus agalactiae, fused to the surface protein of the virus, hemagglutinin, using a flexible linker. Recombinant viruses with ScaAB inserts of 85, 141, and 200 amino acids were successfully rescued by the means of reverse genetics. The recombinant strains were able to grow in developing chicken embryos and MDCK cells and retained the temperature-sensitive phenotype attributable to the LAIV viruses. Studies of immunogenicity and protective activity of the vaccine candidates in BALB/c mice revealed that the most promising strain was a strain with an insert of 141 amino acids: this variant had optimal immunogenicity against influenza and GBS and had a protective effect against both pathogens. These data indicate that further studies of the recombinant vectored vaccine H7-ScaAB-141 as a combined viral-bacterial vaccine capable of protection against both influenza virus and bacterial infections caused by group B streptococci are warranted.

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Identification of Cysteine Residues Essential for von Willebrand Factor (VWF) Multimerization.

Blood ◽

10.1182/blood.v108.11.1799.1799 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1799-1799

Author(s):

Angie R. Purvis ◽

Julia Gross ◽

Luke T. Dang ◽

Ren-Huai Huang ◽

Milan Kapadia ◽

...

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Expression System ◽

Quadrupole Ion Trap ◽

Cysteine Residues ◽

Ion Cyclotron Resonance ◽

Unbound Fraction ◽

Bhk Cells ◽

Interchain Disulfide Bond ◽

Doubly Charged

Abstract VWF assembles into multimers in the Golgi by forming disulfide bonds between adjacent D’D3 domains using an oxidoreductase mechanism that requires the VWF propeptide (D1D2). During multimerization, free thiols are predicted to be important in the oxidoreductase mechanism either as the cysteine residues that form interchain disulfide bonds in the VWF multimer or as the cysteine residues that rearrange the disulfide-bonded intermediate between the propeptide and D’D3. To characterize free thiols involved in VWF multimerization, a model construct containing domains D1D2D’D3 was expressed in baby hamster kidney (BHK) cells and the secreted products were alkylated with thiol-reactive biotin-maleimide reagents and purified on avidin agarose. Both the D’D3 dimer and the D1D2 remained in the avidin agarose unbound fraction, indicating the absence of reduced Cys residues. The D’D3 monomer was bound to the avidin agarose beads, indicating the presence of reduced and alkylated cysteines. The reduced cysteines in the D’D3 monomer were alkylated with N-ethylmaleimide (NEM) for identification by mass spectrometry. NEM-labeled D’D3 monomer was reduced and alkylated with 4-vinylpyridine (4-VP). NEM increases the mass of Cys to 228.05686 Da, while 4-VP shifts the mass of Cys to 208.06703 Da. Peptides were generated with various proteases and analyzed by a linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTMS). A theoretical peptide list was generated including either NEM or 4-VP modification of all Cys residues. The mass spectra were analyzed for m/z signals including up to the triply charged species of the theoretical digest. The MS/MS spectra were searched against a database with MASCOT. Using this method 98% of the 52 Cys residues within the D’D3 region were identified as either 4-VP- or NEM-modified. Cys1142 was identified in a tryptic peptide (ENGYECEWR+NEM, VWF residues 1137–1145) as a doubly charged ion at m/z 655.76031, confirming the previous identification of the Cys1142-Cys1142 interchain disulfide bond linking VWF multimers. A second NEM-modified peptide was identified from an Asp-N protease digest (DCACFC+2 4-VP+1 NEM, residues 1096–1101 of VWF). This species was observed as a doubly charged ion at m/z 498.67584. Analysis of the MS/MS revealed that Cys1099 was NEM-modified, suggesting that it may form an interchain disulfide bond between VWF multimers or may be important in the oxidoreductase mechanism of VWF multimerization. To determine if C1099 and/or C1142 are required to form D’D3 dimers, constructs D1D2D’D3 (C1099A), (C1142A), and (C1099A/C1142A) were expressed in BHK cells and the assembly of D’D3 dimers was assayed by Western blotting. All three mutant VWF proteins were secreted efficiently from BHK cells. However, the levels of D’D3 dimer were reduced markedly in the single mutants when compared with the dimer produced by the wild type D1D2D’D3 expression system. Further, mutation of both C1142 and C1099 (C1099A/C1142A) completely inhibited D’D3 dimerization, evidence that these specific cysteine residues in the D’D3 monomer are involved in the interchain disulfide bonds between VWF multimers and are essential for the oxidoreductase mechanism of VWF multimerization.

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Protective Immunity Based on the Conserved Hemagglutinin Stalk Domain and Its Prospects for Universal Influenza Vaccine Development

BioMed Research International ◽

10.1155/2014/546274 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 24

Author(s):

Madhu Khanna ◽

Sachin Sharma ◽

Binod Kumar ◽

Roopali Rajput

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Natural Infection ◽

Influenza Viruses ◽

Cross Protection ◽

Influenza B ◽

Universal Vaccine ◽

Virus Surface ◽

Head Domain ◽

Globular Head

Influenza virus surface glycoprotein hemagglutinin (HA) is an excellent and chief target thatelicitsneutralizing antibodies during vaccination or natural infection. Its HA2 subunit (stem domain) is most conserved as compared to HA1 subunit (globular head domain). Current influenza vaccine relies on globular head domain that provides protection only against the homologous vaccine strains, rarely provides cross-protection against divergent strains, and needs to be updated annually. There is an urge for a truly universal vaccine that provides broad cross-protection against different subtype influenza A viruses along with influenza B viruses and need not be updated annually. Antibodies against the stem domain of hemagglutinin (HA) are able to neutralize a wide spectrum of influenza virus strains and subtypes. These stem-specific antibodies have great potential for the development of universal vaccine against influenza viruses. In this review, we have discussed the stem-specific cross-reactive antibodies and heterosubtypic protection provided by them. We have also discussed their epitope-based DNA vaccine and their future prospects in this scenario.

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Antibody responses to influenza viruses in paediatric patients and their contacts at the onset of the 2009 pandemic in Mexico

The Journal of Infection in Developing Countries ◽

10.3855/jidc.5052 ◽

2015 ◽

Vol 9 (03) ◽

pp. 259-266 ◽

Cited By ~ 1

Author(s):

Guadalupe Miranda-Novales ◽

Lourdes Arriaga-Pizano ◽

Cristina Herrera-Castillo ◽

Rodolfo Pastelin-Palacios ◽

Nuriban Valero-Pacheco ◽

...

Keyword(s):

Influenza Virus ◽

Antibody Titre ◽

Pandemic Influenza ◽

Swine Influenza ◽

Influenza Viruses ◽

H3n2 Virus ◽

Serological Response ◽

Pandemic Strain ◽

Paediatric Patients ◽

Influenza A H1n1

Introduction: On April 2009, the Mexican Ministry of Health received notification of cases of severe pneumonia mostly affecting young healthy people; this was the beginning of the first influenza pandemic of the 21st century. The nature of the immune response to the influenza A(H1N1)2009 pandemic strain in Mexico at the beginning of the pandemic outbreak has not been completely defined. We describe the serological response to the 2009 pandemic influenza virus in paediatric patients with influenza-like illness, their household contacts (HHCs), and exposed health-care workers (HCWs) at the beginning of the pandemic outbreak in Mexico City. Methodology: thirty pre-epidemic and 129 epidemic samples were collected and serum antibodies were measured against A(H1N1)2009 pandemic virus and two non-pandemic swine influenza viruses by an haemagglutination inhibition assay . Results: 91% (29/32) of the convalescence samples from confirmed patients had an antibody titre ≥ 10 (GMT 25), 63% (41/65) of the HHCs (GMT 12), 41% of HCWs (GMT 6) and 13% (4/30) of pre-epidemic samples (GMT 6) for the pandemic influenza virus. Of the 32 confirmed cases, 60% had an antibody titre ≥ 40 for the pandemic strain, 53% for the A/swine/Iowa(H1N1) virus (GMT 62) and 43% for the A/swine/Texas(H3N2) virus (GMT 66). Conclusion: The antibody response to 2009 pandemic influenza virus was widespread in convalescence samples from patients with confirmed pandemic influenza infection but the GMT was below the protective titre. There was no evidence that antibodies to the swine influenza viruses had cross-protective effect against the 2009 pandemic influenza virus.

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