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1.
J Biol Chem ; 284(39): 26951-63, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19633296

RESUMO

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R(h) 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.


Assuntos
Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Glicosilfosfatidilinositóis/metabolismo , Heparina/análogos & derivados , Heparina/metabolismo , Hepatócitos/imunologia , Hepatócitos/parasitologia , Hepatócitos/patologia , Humanos , Immunoblotting , Modelos Moleculares , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Esporozoítos/metabolismo , Temperatura , Ultracentrifugação
2.
Virology ; 358(2): 273-82, 2007 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-17027056

RESUMO

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.


Assuntos
Encefalite da Califórnia/virologia , Vírus La Crosse/fisiologia , Proteínas Virais de Fusão/fisiologia , Animais , Linhagem Celular , Humanos , Mutagênese , Estrutura Terciária de Proteína/fisiologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Replicação Viral
3.
Virology ; 346(1): 169-79, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16309726

RESUMO

We previously described envelope glycoproteins of an HIV-1 isolate adapted in vitro for growth in microglia that acquired a highly fusogenic phenotype and lower CD4 dependence, as well as resistance to inhibition by anti-CD4 antibodies. Here, we investigated whether similar phenotypic changes are present in vivo. Envelope clones from the brain and spleen of an HIV-1-infected individual with neurological disease were amplified, cloned, and sequenced. Phylogenetic analysis demonstrated clustering of sequences according to the tissue of origin, as expected. Functional clones were then used in cell-to-cell fusion assays to test for CD4 and co-receptor utilization and for sensitivity to various antibodies and inhibitors. Both brain- and spleen-derived envelope clones mediated fusion in cells expressing both CD4 and CCR5 and brain envelopes also used CCR3 as co-receptor. We found that the brain envelopes had a lower CD4 dependence, since they efficiently mediated fusion in the presence of low levels of CD4 on the target cell membrane, and they were significantly more resistant to blocking by anti-CD4 antibodies than the spleen-derived envelopes. In contrast, we observed no difference in sensitivity to the CCR5 antagonist TAK-779. However, brain-derived envelopes were significantly more resistant than those from spleen to the fusion inhibitor T-1249 and concurrently showed slightly greater fusogenicity. Our results suggest an increased affinity for CD4 of brain-derived envelopes that may have originated from in vivo adaptation to replication in microglial cells. Interestingly, we note the presence of envelopes more resistant to a fusion inhibitor in the brain of an untreated, HIV-1-infected individual.


Assuntos
Complexo AIDS Demência/virologia , Encéfalo/virologia , Antígenos CD4/metabolismo , Farmacorresistência Viral , Inibidores da Fusão de HIV/farmacologia , HIV-1/patogenicidade , Sequência de Aminoácidos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Fusão de Membrana , Dados de Sequência Molecular , Receptores de HIV/metabolismo , Baço/virologia
4.
Virology ; 338(1): 121-32, 2005 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-15923017

RESUMO

Members of the California serogroup of orthobunyaviruses, particularly La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness (Janssen, R., Gonzalez-Scarano, F., Nathanson, N., 1984. Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab. Invest. 50 (4), 447-455), have demonstrated that the neuroinvasive phenotype maps to the M segment, the segment that encodes the two viral glycoproteins Gn (G2) and Gc (G1), as well as a non-structural protein NSm. To further define the role of Gn and Gc in fusion and entry, we prepared a panel of recombinant M segment constructs using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were then tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an MLV backbone. Both assays demonstrated that Gc is the principal determinant of virus fusion and cell entry, and furthermore that the region delineated by amino acids 860-1442, corresponding to the membrane proximal two-thirds of Gc, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of Gc and Sindbis virus E1, suggest that the LAC Gc functions as a type II fusion protein.


Assuntos
Vírus da Encefalite da Califórnia/patogenicidade , Vírus La Crosse/patogenicidade , Proteínas Virais/fisiologia , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Vírus da Encefalite da Califórnia/classificação , Vírus da Encefalite da Califórnia/genética , Expressão Gênica , Genes Virais , Humanos , Concentração de Íons de Hidrogênio , Vírus La Crosse/classificação , Vírus La Crosse/genética , Vírus da Leucemia Murina/genética , Modelos Moleculares , Conformação Proteica , Codorniz , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução Genética , Transfecção , Proteínas Virais/química , Proteínas Virais/genética , Virulência
5.
J Virol ; 79(1): 234-44, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15596819

RESUMO

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.


Assuntos
Regulação Viral da Expressão Gênica , Vírus La Crosse/patogenicidade , MicroRNAs/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Vírus La Crosse/genética , Vírus La Crosse/metabolismo , MicroRNAs/genética , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transfecção , Células Vero , Proteínas não Estruturais Virais/genética , Ensaio de Placa Viral , Replicação Viral
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