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1.
PLoS Pathog ; 12(10): e1005813, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27783711

RESUMEN

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.


Asunto(s)
Orthobunyavirus/química , Orthohantavirus/química , Proteínas del Envoltorio Viral/química , Animales , Cristalografía , Glicoproteínas/química , Humanos , Conformación Proteica , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie
2.
J Struct Biol ; 188(1): 71-8, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25172991

RESUMEN

Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Imagen por Resonancia Magnética , Biosíntesis de Proteínas , Proteínas de la Matriz Viral/química , Aminoácidos/química , Animales , Drosophila/química , Drosophila/genética , Humanos , Radioisótopos de Nitrógeno/química , Células Sf9 , Spodoptera
3.
Viruses ; 14(7)2022 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-35891553

RESUMEN

Over the past two years, scientific research has moved at an unprecedented rate in response to the COVID-19 pandemic. The rapid development of effective vaccines and therapeutics would not have been possible without extensive background knowledge on coronaviruses developed over decades by researchers, including Kathryn (Kay) Holmes. Kay's research team discovered the first coronavirus receptors for mouse hepatitis virus and human coronavirus 229E and contributed a wealth of information on coronaviral spike glycoproteins and receptor interactions that are critical determinants of host and tissue specificity. She collaborated with several research laboratories to contribute knowledge in additional areas, including coronaviral pathogenesis, epidemiology, and evolution. Throughout her career, Kay was an extremely dedicated and thoughtful mentor to numerous graduate students and post-doctoral fellows. This article provides a review of her contributions to the coronavirus field and her exemplary mentoring.


Asunto(s)
Coronavirus Humano 229E , Receptores de Coronavirus , Animales , COVID-19 , Historia del Siglo XXI , Humanos , Ratones , Pandemias , Glicoproteína de la Espiga del Coronavirus/genética
4.
J Virol ; 82(6): 2883-94, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18199653

RESUMEN

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Delta19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Delta19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Delta19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.


Asunto(s)
Aminoácidos Aromáticos/fisiología , Fusión Celular , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Receptores Virales/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Aminoácidos Aromáticos/química , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Receptores Virales/química , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/química
6.
Chem Biol Drug Des ; 73(1): 53-61, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19152635

RESUMEN

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel coronavirus that cost nearly 800 lives. While there have been no recent outbreaks of the disease, the threat remains as SARS coronavirus (SARS-CoV) like strains still exist in animal reservoirs. Therefore, the development of a vaccine against SARS is in grave need. Here, we have designed and produced a prototypic SARS vaccine: a self-assembling polypeptide nanoparticle that repetitively displays a SARS B-cell epitope from the C-terminal heptad repeat of the virus' spike protein. Biophysical analyses with circular dichroism, transmission electron microscopy and dynamic light scattering confirmed the computational design showing alpha-helcial nanoparticles with sizes of about 25 nm. Immunization experiments with no adjuvants were performed with BALB/c mice. An investigation of the binding properties of the elicited antibodies showed that they were highly conformation specific for the coiled-coil epitope because they specifically recognized the native trimeric conformation of C-terminal heptad repeat region. Consequently, the antisera exhibited neutralization activity in an in vitro infection inhibition assay. We conclude that these peptide nanoparticles represent a promising platform for vaccine design, in particular for diseases that are characterized by neutralizing epitopes with coiled-coil conformation such as SARS-CoV or other enveloped viruses.


Asunto(s)
Nanopartículas , Péptidos/química , Péptidos/inmunología , Síndrome Respiratorio Agudo Grave/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Vacunas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/química , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Nanopartículas/química , Nanopartículas/ultraestructura , Péptidos/genética , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Síndrome Respiratorio Agudo Grave/inmunología , Vacunas/química
7.
Virology ; 372(1): 127-35, 2008 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-18022664

RESUMEN

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.


Asunto(s)
Epitelio/virología , Alveolos Pulmonares/citología , Alveolos Pulmonares/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Replicación Viral , Diferenciación Celular , Células Cultivadas , Efecto Citopatogénico Viral , Humanos , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Virología/métodos
8.
J Struct Biol ; 155(2): 176-94, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16697221

RESUMEN

The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.


Asunto(s)
Inmunoglobulina G/inmunología , Glicoproteínas de Membrana/inmunología , Péptidos/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Western Blotting/métodos , Células CHO , Dicroismo Circular/métodos , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo , Cinética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/química , Estructura Secundaria de Proteína , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus , Resonancia por Plasmón de Superficie/métodos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética
9.
J Virol ; 76(24): 12463-72, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12438572

RESUMEN

The role of covalent modifications of the Ebola virus glycoprotein (GP) and the significance of the sequence identity between filovirus and avian retrovirus GPs were investigated through biochemical and functional analyses of mutant GPs. The expression and processing of mutant GPs with altered N-linked glycosylation, substitutions for conserved cysteine residues, or a deletion in the region of O-linked glycosylation were analyzed, and virus entry capacities were assayed through the use of pseudotyped retroviruses. Cys-53 was the only GP(1) ( approximately 130 kDa) cysteine residue whose replacement resulted in the efficient secretion of GP(1), and it is therefore proposed that it participates in the formation of the only disulfide bond linking GP(1) to GP(2) ( approximately 24 kDa). We propose a complete cystine bridge map for the filovirus GPs based upon our analysis of mutant Ebola virus GPs. The effect of replacement of the conserved cysteines in the membrane-spanning region of GP(2) was found to depend on the nature of the substitution. Mutations in conserved N-linked glycosylation sites proved generally, with a few exceptions, innocuous. Deletion of the O-linked glycosylation region increased GP processing, incorporation into retrovirus particles, and viral transduction. Our data support a common evolutionary origin for the GPs of Ebola virus and avian retroviruses and have implications for gene transfer mediated by Ebola virus GP-pseudotyped retroviruses.


Asunto(s)
Proteínas del Envoltorio Viral/química , Células 3T3 , Animales , Línea Celular , Glicosilación , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/fisiología
10.
J Virol ; 77(10): 5902-10, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12719583

RESUMEN

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.


Asunto(s)
Proteínas Portadoras/metabolismo , Filoviridae/metabolismo , Vectores Genéticos , Virus de la Inmunodeficiencia Felina/patogenicidad , Receptores de Superficie Celular , Sistema Respiratorio/virología , Transducción Genética , Proteínas del Envoltorio Viral/metabolismo , Animales , Gatos , Polaridad Celular , Células Cultivadas , Células Epiteliales/virología , Filoviridae/genética , Receptores de Folato Anclados a GPI , Humanos , Virus de la Inmunodeficiencia Felina/genética , Virus de la Inmunodeficiencia Felina/metabolismo , Sistema Respiratorio/citología , Proteínas del Envoltorio Viral/genética
11.
Proc Natl Acad Sci U S A ; 101(44): 15748-53, 2004 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-15496474

RESUMEN

Angiotensin-converting enzyme 2 (ACE2) is a receptor for SARS-CoV, the novel coronavirus that causes severe acute respiratory syndrome [Li, W. Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454]. We have identified a different human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV. A human lung cDNA library in vesicular stomatitis virus G pseudotyped retrovirus was transduced into Chinese hamster ovary cells, and the cells were sorted for binding of soluble SARS-CoV spike (S) glycoproteins, S(590) and S(1180). Clones of transduced cells that bound SARS-CoV S glycoprotein were inoculated with SARS-CoV, and increases in subgenomic viral RNA from 1-16 h or more were detected by multiplex RT-PCR in four cloned cell lines. Sequencing of the human lung cDNA inserts showed that each of the cloned cell lines contained cDNA that encoded human CD209L, a C-type lectin (also called L-SIGN). When the cDNA encoding CD209L from clone 2.27 was cloned and transfected into Chinese hamster ovary cells, the cells expressed human CD209L glycoprotein and became susceptible to infection with SARS-CoV. Immunohistochemistry showed that CD209L is expressed in human lung in type II alveolar cells and endothelial cells, both potential targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of entry, and HIV and hepatitis C virus can bind to CD209L on cell membranes but do not use it to mediate virus entry. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de la Membrana/fisiología , Receptores Virales/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Animales , Secuencia de Bases , Células CHO , Proteínas de Ciclo Celular/genética , Línea Celular , Cricetinae , ADN Complementario/genética , Biblioteca de Genes , Humanos , Pulmón/metabolismo , Pulmón/virología , Proteínas de la Membrana/genética , Receptores de Coronavirus , Receptores Virales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Transducción Genética
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