scholarly journals Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

2005 ◽  
Vol 79 (10) ◽  
pp. 6449-6458 ◽  
Author(s):  
Ralf Wagner ◽  
Astrid Herwig ◽  
Nahid Azzouz ◽  
Hans Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Wild Type Virus ◽  
Fusion Pore ◽  
Cysteine Residues ◽  
Virus Infectivity ◽  
Wild Type ◽  
Protein Patterns ◽  
Virus Growth ◽  
Influenza Virus Hemagglutinin ◽  
Fusion Pores

ABSTRACT Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity.

scholarly journals Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

1999 ◽  
Vol 10 (6) ◽  
pp. 1821-1836 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sasa Lin ◽  
Michael G. Roth ◽  
Fredric S. Cohen
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Membrane Fusion ◽  
Integral Membrane Protein ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Tm Domain ◽  
Fusion Pores ◽  
Sequence Requirements

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

scholarly journals Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

2000 ◽  
Vol 74 (16) ◽  
pp. 7529-7537 ◽  
Author(s):  
Christine Kozerski ◽  
Evgeni Ponimaskin ◽  
Britta Schroth-Diez ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Expression System ◽  
Fusion Pore ◽  
Target Cells ◽  
Fusion Activity ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Pore Enlargement

ABSTRACT The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

scholarly journals Design and Structure of an Engineered Disulfide-Stabilized Influenza Virus Hemagglutinin Trimer

2015 ◽  
Vol 89 (14) ◽  
pp. 7417-7420 ◽  
Author(s):  
Peter S. Lee ◽  
Xueyong Zhu ◽  
Wenli Yu ◽  
Ian A. Wilson
Keyword(s):  
Influenza Virus ◽  
Structural Changes ◽  
Cysteine Residues ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin

We engineered a disulfide-stabilized influenza virus hemagglutinin (HA) trimer, termed HA3-SS, by introducing cysteine residues into the HA stem to covalently bridge the three protomers. HA3-SS has increased thermostability compared to wild-type HA, and binding of head- and stem-targeted antibodies (Abs) is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza virus HA immunogens.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

scholarly journals Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

1998 ◽  
Vol 42 (12) ◽  
pp. 3234-3241 ◽  
Author(s):  
Chun Y. Tai ◽  
Paul A. Escarpe ◽  
Robert W. Sidwell ◽  
Matthew A. Williams ◽  
Willard Lew ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Neuraminidase Inhibitor ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Human Influenza ◽  
Wild Type ◽  
Viral Virulence ◽  
High Level

ABSTRACT An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Dual Wavelength Imaging Allows Analysis of Membrane Fusion of Influenza Virus inside Cells

2006 ◽  
Vol 80 (4) ◽  
pp. 2013-2018 ◽  
Author(s):  
Tatsuya Sakai ◽  
Masanobu Ohuchi ◽  
Masaki Imai ◽  
Takafumi Mizuno ◽  
Kazunori Kawasaki ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Imaging Technique ◽  
Rapid Analysis ◽  
Virus Infectivity ◽  
Viral Fusion ◽  
The Novel ◽  
Influenza Virus Hemagglutinin ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

ABSTRACT Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.

scholarly journals Computationally Optimized Broadly Reactive H2 HA Influenza Vaccines Elicited Broadly Cross-Reactive Antibodies and Protected Mice from Viral Challenges

2020 ◽  
Vol 95 (2) ◽  
pp. e01526-20
Author(s):  
Z. Beau Reneer ◽  
Parker J. Jamieson ◽  
Amanda L. Skarlupka ◽  
Ying Huang ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Influenza Vaccine ◽  
Neutralizing Antibodies ◽  
Influenza Pandemic ◽  
Mammalian Species ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Wild Type ◽  
The 1950S ◽  
Future Work

ABSTRACTInfluenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of hemagglutinin (HA) antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this study, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) was evaluated in DBA/2J mice and compared to that of wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by hemagglutination inhibition (HAI) and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work toward a pandemic H2 influenza vaccine.IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.

scholarly journals Inactivation of hantaviruses by N-ethylmaleimide preserves virion integrity

2011 ◽  
Vol 92 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Tomas Strandin ◽  
Jussi Hepojoki ◽  
Hao Wang ◽  
Antti Vaheri ◽  
Hilkka Lankinen
Keyword(s):  
Cell Attachment ◽  
Buoyant Density ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Thiol Groups ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Nitrobenzoic Acid ◽  
The Family

Thiol groups of cysteine residues are crucial for the infectivity of various enveloped viruses, but their role in the infectivity of viruses of the family Bunyaviridae has thus far not been studied. This report shows that thiol groups are essential to the infectivity of hantaviruses. Alkylation of the thiol functional groups using the membrane-permeable compound N-ethylmaleimide (NEM) and membrane-impermeable compound 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) showed NEM to be a highly effective inactivator of Puumala and Tula hantaviruses. The NEM-inactivated hantavirus maintained the buoyant density of the wild-type virus. Furthermore, the antigenicity of glycoproteins and the cell attachment capacity of virions were retained at NEM concentrations that totally abolished virus infectivity. These results signified preservation of virion integrity following inactivation with NEM, making chemically inactivated virions valuable research antigens. It was demonstrated with biotin-conjugated maleimide, a mechanistic analogue of NEM, that all the structural proteins of hantavirus were sensitive towards thiol alkylation. In contrast to hantaviruses, NEM did not abolish Uukuniemi phlebovirus infectivity to the same extent. This indicates differences in the use of free thiols in virus entry among members of the family Bunyaviridae.

scholarly journals A Point Mutation in the Transmembrane Domain of the Hemagglutinin of Influenza Virus Stabilizes a Hemifusion Intermediate That Can Transit to Fusion

2000 ◽  
Vol 11 (11) ◽  
pp. 3765-3775 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Ruben M. Markosyan ◽  
Michael G. Roth ◽  
Fredric S. Cohen
Keyword(s):  
Influenza Virus ◽  
Point Mutation ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Wild Type ◽  
Decreasing Ph ◽  
Lower Temperature ◽  
Increasing Temperature ◽  
Fusion Pores

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

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