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1.
Virology ; 514: 216-229, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29202415

RESUMEN

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune-modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin-JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease.


Asunto(s)
Fiebre Hemorrágica Americana/inmunología , Virus Junin/fisiología , Gotas Lipídicas/virología , Proteínas/inmunología , Fiebre Hemorrágica Americana/genética , Fiebre Hemorrágica Americana/virología , Humanos , Interferones/genética , Interferones/inmunología , Virus Junin/química , Virus Junin/genética , Virus Junin/inmunología , Gotas Lipídicas/inmunología , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Dominios Proteicos , Proteínas/química , Proteínas/genética , Replicación Viral
2.
Cell Host Microbe ; 18(6): 705-13, 2015 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-26651946

RESUMEN

In the Western hemisphere, at least five mammarenaviruses cause human viral hemorrhagic fevers with high case fatality rates. Junín virus (JUNV) is the only hemorrhagic fever virus for which transfusion of survivor immune plasma that contains neutralizing antibodies ("passive immunity") is an established treatment. Here, we report the structure of the JUNV surface glycoprotein receptor-binding subunit (GP1) bound to a neutralizing monoclonal antibody. The antibody engages the GP1 site that binds transferrin receptor 1 (TfR1)-the host cell surface receptor for all New World hemorrhagic fever mammarenaviruses-and mimics an important receptor contact. We show that survivor immune plasma contains antibodies that bind the same epitope. We propose that viral receptor-binding site accessibility explains the success of passive immunity against JUNV and that this functionally conserved epitope is a potential target for therapeutics and vaccines to limit infection by all New World hemorrhagic fever mammarenaviruses.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Virus Junin/química , Glicoproteínas de Membrana/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Cristalografía por Rayos X , Humanos , Virus Junin/inmunología , Glicoproteínas de Membrana/inmunología , Unión Proteica , Conformación Proteica
3.
J Gen Virol ; 94(Pt 10): 2175-2183, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23884367

RESUMEN

Junin virus (JUNV) has been identified as the aetiological agent of Argentine haemorrhagic fever (AHF), which is a serious public health problem with approximately 5 million people at risk. It is treated as a potential bioterrorism agent because of its rapid transmission by aerosols. JUNV is a negative-sense ssRNA virus that belongs to the genus Arenavirus within the family Arenaviridae, and its genomic RNA contains two segments encoding four proteins. Among these, the nucleoprotein (NP) has essential roles in viral RNA synthesis and immune suppression, but the molecular mechanisms of its actions are only partially understood. Here, we determined a 2.2 Å crystal structure of the C-terminal domain of JUNV NP. This structure showed high similarity to the Lassa fever virus (LASV) NP C-terminal domain. However, both the structure and function of JUNV NP showed differences compared with LASV NP. This study extends our structural insight into the negative-sense ssRNA virus NPs.


Asunto(s)
Virus Junin/química , Virus Junin/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Secuencia de Aminoácidos , Regulación Viral de la Expresión Génica/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia
4.
J Biol Chem ; 286(2): 1528-36, 2011 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-21068387

RESUMEN

Arenaviruses cause acute hemorrhagic fevers with high mortality. Entry of the virus into the host cell is mediated by the viral envelope glycoprotein, GPC. In contrast to other class I viral envelope glycoproteins, the mature GPC complex contains a cleaved stable signal peptide (SSP) in addition to the canonical receptor-binding (G1) and transmembrane fusion (G2) subunits. SSP is critical for intracellular transport of the GPC complex to the cell surface and for its membrane-fusion activity. Previous studies have suggested that SSP is retained in GPC through interaction with a zinc-binding domain (ZBD) in the cytoplasmic tail of G2. Here we used NMR spectroscopy to determine the structure of Junín virus (JUNV) ZBD (G2 residues 445-485) and investigate its interaction with a conserved Cys residue (Cys-57) in SSP. We show that JUNV ZBD displays a novel fold containing two zinc ions. One zinc ion is coordinated by His-447, His-449, Cys-455, and His-485. The second zinc ion is coordinated by His-459, Cys-467, and Cys-469 and readily accepts Cys-57 from SSP as the fourth ligand. Our studies describe the structural basis for retention of the unique SSP subunit and suggest a mechanism whereby SSP is positioned in the GPC complex to modulate pH-dependent membrane fusion.


Asunto(s)
Virus Junin/química , Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Zinc/química , Secuencia de Aminoácidos , Secuencia Conservada , Concentración de Iones de Hidrógeno , Virus Junin/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Señales de Clasificación de Proteína/fisiología , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Zinc/metabolismo , Dedos de Zinc/fisiología
5.
J Virol ; 84(12): 6119-29, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20392854

RESUMEN

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.


Asunto(s)
Anticuerpos/farmacología , Glicoproteínas/inmunología , Virus Junin/fisiología , Fusión de Membrana/efectos de los fármacos , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/virología , Chlorocebus aethiops , Glicoproteínas/química , Glicoproteínas/genética , Concentración de Iones de Hidrógeno , Virus Junin/química , Virus Junin/efectos de los fármacos , Virus Junin/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Células Vero , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética
6.
Antiviral Res ; 84(1): 31-7, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19591878

RESUMEN

Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179-197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.


Asunto(s)
Infecciones por Arenaviridae/virología , Regulación hacia Abajo , Virus Junin/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Replicación Viral , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cricetinae , Virus Junin/química , Virus Junin/fisiología , Datos de Secuencia Molecular , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Células Vero
7.
J Virol ; 81(24): 13385-91, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17928348

RESUMEN

The envelope glycoprotein of the Junín arenavirus (GP-C) mediates entry into target cells through a pH-dependent membrane fusion mechanism. Unlike other class I viral fusion proteins, the mature GP-C complex retains a cleaved, 58-amino-acid signal peptide (SSP) as an essential subunit, required both for trafficking of GP-C to the cell surface and for the activation of membrane fusion. SSP has been shown to associate noncovalently in GP-C via the cytoplasmic domain (CTD) of the transmembrane fusion subunit G2. In this report we investigate the molecular basis for this intersubunit interaction. We identify an invariant series of six cysteine and histidine residues in the CTD of G2 that is essential for incorporation of SSP in the GP-C complex. Moreover, we show that a CTD peptide fragment containing His-447, His-449, and Cys-455 specifically binds Zn(2+) at subnanomolar concentrations. Together, these results suggest a zinc finger-like domain structure in the CTD of G2. We propose that the remaining residues in the series (His-459, Cys-467, and Cys-469) form an intersubunit zinc-binding center that incorporates Cys-57 of SSP. This unusual motif may act to retain SSP in the GP-C complex and position the ectodomain loop of SSP for its role in modulating membrane fusion activity. The unique tripartite organization of GP-C could provide novel molecular targets for therapeutic intervention in arenaviral disease.


Asunto(s)
Glicoproteínas/metabolismo , Virus Junin/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/metabolismo , Zinc/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Glicoproteínas/química , Glicoproteínas/genética , Concentración de Iones de Hidrógeno , Virus Junin/química , Virus Junin/genética , Fusión de Membrana , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Dedos de Zinc/genética
8.
J Virol ; 81(8): 4331-7, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17267481

RESUMEN

The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hphi1 and hphi2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hphi2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.


Asunto(s)
Membrana Celular/química , Membrana Celular/virología , Citoplasma/química , Virus Junin/química , Señales de Clasificación de Proteína , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Fusión Celular , Detergentes/farmacología , Digitonina/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Virus Junin/fisiología , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus/fisiología
9.
J Virol ; 80(11): 5189-98, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16698999

RESUMEN

Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.


Asunto(s)
Glicoproteínas/metabolismo , Virus Junin/química , Señales de Clasificación de Proteína/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Transporte Biológico , Chlorocebus aethiops , Glicoproteínas/genética , Estructura Terciaria de Proteína , Células Vero , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo
10.
J Virol ; 78(19): 10783-92, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15367645

RESUMEN

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.


Asunto(s)
Glicoproteínas/química , Virus Junin/química , Señales de Clasificación de Proteína/fisiología , Subunidades de Proteína/metabolismo , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos CD4/genética , Antígenos CD4/metabolismo , Fusión Celular , Chlorocebus aethiops , Secuencia Conservada , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Fusión de Membrana , Datos de Secuencia Molecular , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo
11.
Virus Res ; 73(1): 41-55, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11163643

RESUMEN

RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.


Asunto(s)
Arenavirus/fisiología , Células Eucariotas/virología , Proteínas de la Nucleocápside/fisiología , Animales , Arenavirus/genética , Arenavirus/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Cricetinae , Virus Junin/química , Virus Junin/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Transcripción Genética , Activación Transcripcional , Transfección , Replicación Viral/genética
12.
Virology ; 272(1): 127-36, 2000 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-10873755

RESUMEN

RNA was purified from 39 strains of cell-cultured Junin virus (JUN) from central Argentina, which included both human- and rodent-derived isolates (a total of 26 and 13, respectively), as well as from 2 laboratory JUN strains, XJ Cl3 and XJ #44. JUN-specific primers were used to amplify a 511-nucleotide (nt) fragment of the nucleocapsid protein gene and a 495-nt fragment of the glycoprotein 1 (GP1) gene. Genetic diversity among JUN strains studied was up to 13% at the nt level and up to 9% at the amino acid (aa) level for the GP1 gene and up to 9% (nt) and 4% (aa) for the NP gene. Phylogenetic analyses of both genes revealed three distinct clades. The first clade was composed of the JUN strains from the center of the endemic area and included the majority of JUN strains analyzed in the current study. The second clade contained 4 JUN strains isolated between 1963 and 1971 from Cordoba Province, the western-most edge of the known endemic area. The third clade contained 4 JUN strains that originated from Calomys musculinus trapped in Zarate, the northeastern edge of the known endemic area. Certain JUN sequences, which were obtained from GenBank and identified as XJ, XJ #44, and Candid #1 strains, appeared to form a separate clade. Over 400 nt of the GP1 and GP2 genes were additionally sequenced for 7 JUN strains derived from patients with different clinical presentations and outcomes of Argentine hemorrhagic fever. Analysis of the corresponding aa sequences did not allow us to attribute any particular genetic marker to the changing severity or clinical form of the human disease.


Asunto(s)
Variación Genética/genética , Fiebre Hemorrágica Americana/epidemiología , Fiebre Hemorrágica Americana/virología , Virus Junin/clasificación , Virus Junin/genética , Filogenia , Animales , Argentina/epidemiología , Línea Celular , Análisis Mutacional de ADN , Genes Virales/genética , Glicoproteínas/química , Glicoproteínas/genética , Fiebre Hemorrágica Americana/fisiopatología , Humanos , Virus Junin/química , Virus Junin/patogenicidad , Ratones , Datos de Secuencia Molecular , Muridae/virología , Mutación/genética , Nucleocápside/química , Nucleocápside/genética , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Factores de Tiempo , Virulencia/genética
13.
J Virol Methods ; 80(2): 217-21, 1999 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10471032

RESUMEN

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.


Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Glicoproteínas/química , Glicósidos/metabolismo , Proteínas Virales/química , Animales , Chlorocebus aethiops , Glicósido Hidrolasas/metabolismo , Radioisótopos de Yodo/metabolismo , Virus Junin/química , Virus Junin/aislamiento & purificación , Mapeo Peptídico/métodos , Virus de la Rubéola/química , Virus de la Rubéola/aislamiento & purificación , Células Vero
14.
Am J Trop Med Hyg ; 56(2): 216-25, 1997 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9080883

RESUMEN

A live attenuated virus vaccine has been recently developed to prevent Argentine hemorrhagic fever. In this paper, we report the nucleotide sequence of the glycoprotein precursor gene (GPC) of the Junin virus vaccine strain (Candid #1) and its flanking untranslated regions. The untranslated regions flanking the GPC genes of different arenaviruses are variable in length, sequence, and secondary structure. However, when this highly attenuated Junin virus strain is compared with the MC2 strain, which is of intermediate virulence, one nucleotide insertion and four nucleotide substitutions are found at positions that do not affect the predicted secondary structure. When Candid #1 and MC2 RNAs are compared, the nucleotide sequence changes in the GPC open reading frame are concentrated in the amino-proximal and the carboxy-proximal regions. The comparison of the amino acid residues shows that the major changes are located in the amino-proximal region of the GPC.


Asunto(s)
Glicoproteínas/genética , Virus Junin/genética , Precursores de Proteínas/genética , ARN Viral/química , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Viral/química , Glicoproteínas/química , Humanos , Virus Junin/química , Datos de Secuencia Molecular , Precursores de Proteínas/química , Estructura Secundaria de Proteína , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Vacunas Atenuadas/química , Vacunas Atenuadas/genética , Proteínas Estructurales Virales/química , Vacunas Virales/química , Vacunas Virales/genética
15.
J Virol Methods ; 63(1-2): 27-35, 1997 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9015273

RESUMEN

Results suggest that protein, glycerophospolipid, galactoside, and sialyl glycoside residues are present in Junin virus (JV), are accessible to enzymatic digestion, and play an important role in infection. Four major protein bands with molecular masses (Mr) 64 +/- 2, 56 +/- 2, 52 +/- 3 (mean +/- standard deviation, n = 4) and approximately 12-18 kDa were consistently detected after denaturing gel electrophoresis analysis of purified attenuated JV. The 52 kDa protein showed a diffuse tail in the 52-56 kDa range and was considered to be the JV glycoprotein. By Western blotting, the 64 kDa protein bound a JV neutralizing antibody and was considered to be the viral nucleoprotein. Additional bands corresponding to larger proteins (approximately 200, 96, 86, and 78 80 kDa in size), as well as fainter and broader bands in the 23-44 kDa region were also present in purified JV preparations. The relative resistance of virus infectivity to RNase digestion demonstrates that the genome of JV is protected from enzymatic attack. Analysis of purified JV virions by electrophoresis resolved the viral small (S) RNA and large (L) RNA species, 3636 +/- 54 bases and 7667 +/- 154 bases long, respectively (average length +/- range, in four determinations). The (S) RNA of attenuated JV appeared slightly larger than that reported for a pathogenic strain, ruling out a large sequence deletion as a reason for attenuation.


Asunto(s)
Virus Junin/genética , Animales , Chlorocebus aethiops , Humanos , Virus Junin/química , Virus Junin/patogenicidad , ARN Viral/análisis , Células Vero , Proteínas Virales/análisis , Proteínas Virales/genética
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