scholarly journals Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

2000 ◽  
Vol 74 (14) ◽  
pp. 6316-6323 ◽  
Author(s):  
Ralf Wagner ◽  
Thorsten Wolff ◽  
Astrid Herwig ◽  
Stephan Pleschka ◽  
Hans-Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Simian Virus 40 ◽  
Host Cells ◽  
Receptor Binding Site ◽  
Virus Growth ◽  
Recombinant Viruses

ABSTRACT The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

scholarly journals Infectivity Studies of Influenza Virus Hemagglutinin Receptor Binding Site Mutants in Mice

2008 ◽  
Vol 82 (10) ◽  
pp. 5079-5083 ◽  
Author(s):  
Jeffrey Meisner ◽  
Kristy J. Szretter ◽  
Konrad C. Bradley ◽  
William A. Langley ◽  
Zhu-Nan Li ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Binding Site ◽  
Receptor Binding ◽  
Mdck Cells ◽  
Receptor Binding Site ◽  
Binding Characteristics ◽  
Influenza Virus Hemagglutinin ◽  
Fold Reduction ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.

scholarly journals Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors

2005 ◽  
Vol 79 (19) ◽  
pp. 12416-12424 ◽  
Author(s):  
Vasiliy P. Mishin ◽  
Dmitri Novikov ◽  
Frederick G. Hayden ◽  
Larisa V. Gubareva
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Dominant Role ◽  
Neuraminidase Inhibitors ◽  
Virus Binding ◽  
Receptor Binding Site ◽  
Virus Susceptibility ◽  
Infected Cells

ABSTRACT Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA activity for replication. With the use of reverse genetics technique, we investigated the effect of glycans attached at Asn 94a, 129, and 163 on the virus susceptibility to NA inhibitors in MDCK cells and demonstrated that the glycan attached at Asn 163 plays a dominant role in compensation for the loss of NA activity.

scholarly journals Inactivation of influenza virus haemagglutinin by chlorine dioxide: oxidation of the conserved tryptophan 153 residue in the receptor-binding site

2012 ◽  
Vol 93 (12) ◽  
pp. 2558-2563 ◽  
Author(s):  
Norio Ogata
Keyword(s):  
Influenza Virus ◽  
Binding Site ◽  
Receptor Binding ◽  
Chlorine Dioxide ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Life Time ◽  
Tryptophan Residue ◽  
Dependent Manner ◽  
Receptor Binding Site

Airborne influenza virus infection of mice can be prevented by gaseous chlorine dioxide (ClO2). This study demonstrated that ClO2 abolished the function of the haemagglutinin (HA) of influenza A virus (H1N1) in a concentration-, time- and temperature-dependent manner. The IC50 during a 2 min reaction with ClO2 at 25 °C was 13.7 µM, and the half-life time of HA with 100 µM ClO2 at 25 °C was 19.5 s. Peptides generated from a tryptic digest of ClO2-treated virus were analysed by mass spectrometry. An HA fragment, 150NLLWLTGK157 was identified in which the tryptophan residue (W153) was 32 mass units greater than expected. The W153 residue of this peptide, which is derived from the central region of the receptor-binding site of HA, is highly conserved. It was shown that W153 was oxidized to N-formylkynurenine in ClO2-treated virus. It was concluded that the inactivation of influenza virus by ClO2 is caused by oxidation of W153 in HA, thereby abolishing its receptor-binding ability.

scholarly journals Neuraminidase Inhibitor Sensitivity and Receptor-Binding Specificity of Cambodian Clade 1 Highly Pathogenic H5N1 Influenza Virus

2011 ◽  
Vol 55 (5) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Naughtin ◽  
J. C. Dyason ◽  
S. Mardy ◽  
S. Sorn ◽  
M. von Itzstein ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Binding Specificity ◽  
Neuraminidase Inhibitor ◽  
H5n1 Influenza Virus ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Receptor Binding Specificity ◽  
Pathogenic H5n1

ABSTRACTThe evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

scholarly journals Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Irina Baranovskaya ◽  
Mariia Sergeeva ◽  
Artem Fadeev ◽  
Renata Kadirova ◽  
Anna Ivanova ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Secondary Structure ◽  
Protein Expression ◽  
Rna Secondary Structure ◽  
Reverse Genetics ◽  
Early Stage ◽  
Influenza Virus Infection ◽  
Main Function ◽  
Ns1 Protein ◽  
Virus Strains

AbstractRNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

scholarly journals Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

1996 ◽  
Vol 40 (1) ◽  
pp. 40-46 ◽  
Author(s):  
J L McKimm-Breschkin ◽  
T J Blick ◽  
A Sahasrabudhe ◽  
T Tiong ◽  
D Marshall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Receptor Binding ◽  
Liquid Culture ◽  
Plaque Assay ◽  
Enoic Acid ◽  
Cross Resistance ◽  
Receptor Binding Site ◽  
Significant Difference

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

scholarly journals User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 301
Author(s):  
Bingyu Yan ◽  
Xiaohui Zou ◽  
Xinglong Liu ◽  
Jiaming Zhao ◽  
Wenfeng Zhang ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Economic Losses ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Fowl Adenovirus ◽  
Viral Genes ◽  
Lmh Cells ◽  
Species Specific ◽  
The Right ◽  
User Friendly

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

scholarly journals Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

2010 ◽  
Vol 84 (8) ◽  
pp. 3730-3737 ◽  
Author(s):  
Jacqueline P. Upham ◽  
Danielle Pickett ◽  
Tatsuro Irimura ◽  
E. Margot Anders ◽  
Patrick C. Reading
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Viral Entry ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Mannose Receptor ◽  
Host Cells ◽  
Cell Type ◽  
Carbohydrate Recognition Domains ◽  
Macrophage Populations

ABSTRACT Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca2+-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.

scholarly journals Improvement of Influenza A/Fujian/411/02 (H3N2) Virus Growth in Embryonated Chicken Eggs by Balancing the Hemagglutinin and Neuraminidase Activities, Using Reverse Genetics

2005 ◽  
Vol 79 (11) ◽  
pp. 6763-6771 ◽  
Author(s):  
Bin Lu ◽  
Helen Zhou ◽  
Dan Ye ◽  
George Kemble ◽  
Hong Jin
Keyword(s):  
Amino Acids ◽  
Virus Replication ◽  
Receptor Binding ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Vaccines ◽  
H3n2 Virus ◽  
Virus Growth ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.

scholarly journals Reverse Genetic Generation of Recombinant Zaire Ebola Viruses Containing Disrupted IRF-3 Inhibitory Domains Results in Attenuated Virus Growth In Vitro and Higher Levels of IRF-3 Activation without Inhibiting Viral Transcription or Replication

2006 ◽  
Vol 80 (13) ◽  
pp. 6430-6440 ◽  
Author(s):  
Amy L. Hartman ◽  
Jason E. Dover ◽  
Jonathan S. Towner ◽  
Stuart T. Nichol
Keyword(s):  
Reverse Genetics ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Type I ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Inhibitory Function ◽  
C Terminus ◽  
Inhibitory Domain

ABSTRACT The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-β production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

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