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2.
Front Microbiol ; 12: 708480, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335544

RESUMO

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40 T . We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

3.
Nat Commun ; 12(1): 4320, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262046

RESUMO

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Norovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Variação Antigênica , Sítios de Ligação , Antígenos de Grupos Sanguíneos/metabolismo , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/metabolismo , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Mapeamento de Epitopos , Genótipo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Norovirus/genética , Ligação Proteica , Domínios Proteicos
4.
Nat Commun ; 12(1): 4158, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230497

RESUMO

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.


Assuntos
Cicloexenos/síntese química , Cicloexenos/metabolismo , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/metabolismo , Vias Biossintéticas/genética , Catálise , Técnicas de Química Sintética , Compostos de Epóxi , Fermentação , Proteínas Fúngicas/genética , Modelos Moleculares , Estrutura Molecular , Oxigenases , Penicillium/genética , Penicillium/metabolismo
5.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626675

RESUMO

Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.


Assuntos
Arginina/metabolismo , Domínio Catalítico/genética , Histidina/metabolismo , Norovirus/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Infecções por Caliciviridae/metabolismo , Domínio Catalítico/fisiologia , Variação Genética/genética , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Norovirus/genética , Filogenia , Proteólise
6.
Nat Commun ; 9(1): 5010, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30479342

RESUMO

Neonatal rotavirus infections are predominantly asymptomatic. While an association with gastrointestinal symptoms has been described in some settings, factors influencing differences in clinical presentation are not well understood. Using multidisciplinary approaches, we show that a complex interplay between human milk oligosaccharides (HMOs), milk microbiome, and infant gut microbiome impacts neonatal rotavirus infections. Validating in vitro studies where HMOs are not decoy receptors for neonatal strain G10P[11], population studies show significantly higher levels of Lacto-N-tetraose (LNT), 2'-fucosyllactose (2'FL), and 6'-siallylactose (6'SL) in milk from mothers of rotavirus-positive neonates with gastrointestinal symptoms. Further, these HMOs correlate with abundance of Enterobacter/Klebsiella in maternal milk and infant stool. Specific HMOs also improve the infectivity of a neonatal strain-derived rotavirus vaccine. This study provides molecular and translational insight into host factors influencing neonatal rotavirus infections and identifies maternal components that could promote the performance of live, attenuated rotavirus vaccines.


Assuntos
Microbioma Gastrointestinal , Leite Humano/química , Leite Humano/microbiologia , Oligossacarídeos/metabolismo , Infecções por Rotavirus/microbiologia , Fezes/microbiologia , Humanos , Recém-Nascido , Rotavirus/patogenicidade , Infecções por Rotavirus/imunologia , Vacinas contra Rotavirus/imunologia
7.
Curr Opin Struct Biol ; 44: 211-218, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28591681

RESUMO

Recognition and binding to host glycans present on cellular surfaces is an initial and critical step in viral entry. Diverse families of host glycans such as histo-blood group antigens, sialoglycans and glycosaminoglycans are recognized by viruses. Glycan binding determines virus-host specificity, tissue tropism, pathogenesis and potential for interspecies transmission. Viruses including noroviruses, rotaviruses, enteroviruses, influenza, and papillomaviruses have evolved novel strategies to bind specific glycans often in a strain-specific manner. Structural studies have been instrumental in elucidating the molecular determinants of these virus-glycan interactions, aiding in developing vaccines and antivirals targeting this key interaction. Our review focuses on these key structural aspects of virus-glycan interactions, particularly highlighting the different strain-specific strategies employed by viruses to bind host glycans.


Assuntos
Interações Hospedeiro-Patógeno , Polissacarídeos/metabolismo , Fenômenos Fisiológicos Virais , Vírus/metabolismo , Animais , Humanos
8.
Cell Mol Gastroenterol Hepatol ; 2(3): 263-273, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-28090561

RESUMO

Interaction with cellular glycans is a critical initial step in the pathogenesis of many infectious agents. Technological advances in glycobiology have expanded the repertoire of studies delineating host glycan-pathogen interactions. For rotavirus, the VP8* domain of the outer capsid spike protein VP4 is known to interact with cellular glycans. Sialic acid was considered the key cellular attachment factor for rotaviruses for decades. Although this is true for many rotavirus strains causing infections in animals, glycan array screens show that many human rotavirus strains bind nonsialylated glycoconjugates, called histo-blood group antigens, in a strain-specific manner. The expression of histo-blood group antigens is determined genetically and is regulated developmentally. Variations in glycan binding between different rotavirus strains are biologically relevant and provide new insights into multiple aspects of virus pathogenesis such as interspecies transmission, host range restriction, and tissue tropism. The genetics of glycan expression may affect susceptibility to different rotavirus strains and vaccine viruses, and impact the efficacy of rotavirus vaccination in different populations. A multidisciplinary approach to understanding rotavirus-host glycan interactions provides molecular insights into the interaction between microbial pathogens and glycans, and opens up new avenues to translate findings from the bench to the human population.

9.
Nat Commun ; 6: 8346, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26420502

RESUMO

Strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. Such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.


Assuntos
Doenças dos Bovinos/virologia , Polissacarídeos/química , Vírus Reordenados/metabolismo , Receptores Virais/química , Infecções por Rotavirus/metabolismo , Infecções por Rotavirus/veterinária , Rotavirus/metabolismo , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Gastroenterite/metabolismo , Gastroenterite/virologia , Humanos , Polissacarídeos/metabolismo , Vírus Reordenados/química , Vírus Reordenados/genética , Receptores Virais/metabolismo , Rotavirus/química , Rotavirus/genética , Infecções por Rotavirus/virologia , Especificidade da Espécie , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
11.
Curr Opin Virol ; 7: 119-27, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25073118

RESUMO

A critical event in the life cycle of a virus is its initial attachment to host cells. This involves recognition by the viruses of specific receptors on the cell surface, including glycans. Viruses typically exhibit strain-dependent variations in recognizing specific glycan receptors, a feature that contributes significantly to cell tropism, host specificity, host adaptation and interspecies transmission. Examples include influenza viruses, noroviruses, rotaviruses, and parvoviruses. Both rotaviruses and noroviruses are well known gastroenteric pathogens that are of significant global health concern. While rotaviruses, in the family Reoviridae, are the major causative agents of life-threatening diarrhea in children, noroviruses, which belong to the Caliciviridae family, cause epidemic and sporadic cases of acute gastroenteritis across all age groups. Both exhibit enormous genotypic and serotypic diversity. Consistent with this diversity each exhibits strain-dependent variations in the types of glycans they recognize for cell attachment. This chapter reviews the current status of the structural biology of such strain-dependent glycan specificities in these two families of viruses.


Assuntos
Gastroenterite/metabolismo , Norovirus/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Rotavirus/metabolismo , Animais , Gastroenterite/virologia , Humanos , Norovirus/genética , Rotavirus/genética , Especificidade da Espécie
12.
J Virol ; 87(9): 4818-25, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23408637

RESUMO

The major capsid protein of norovirus VP1 assembles to form an icosahedral viral particle. Despite evidence that the Norwalk virus (NV) minor structural protein VP2 is present in infectious virions, the available crystallographic and electron cryomicroscopy structures of NV have not revealed the location of VP2. In this study, we determined that VP1 associates with VP2 at the interior surface of the capsid, specifically with the shell (S) domain of VP1. We mapped the interaction site to amino acid 52 of VP1, an isoleucine located within a sequence motif IDPWI in the S domain that is highly conserved across norovirus genogroups. Mutation of this isoleucine abrogated VP2 incorporation into virus-like particles without affecting the ability for VP1 to dimerize and form particles. The highly basic nature of VP2 and its location interior to the viral particle are consistent with its potential role in assisting capsid assembly and genome encapsidation.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus Norwalk/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Regulação Viral da Expressão Gênica , Humanos , Dados de Sequência Molecular , Vírus Norwalk/química , Vírus Norwalk/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência
13.
Antimicrob Agents Chemother ; 56(11): 5667-77, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22908171

RESUMO

Metallo-ß-lactamases catalyze the hydrolysis of a broad range of ß-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ß-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ß-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ß-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 µM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.


Assuntos
Cisteína/metabolismo , Escherichia coli/genética , Glicina/metabolismo , Zinco/química , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Cisteína/química , Cisteína/genética , Escherichia coli/enzimologia , Glicina/química , Glicina/genética , Cinética , Ligantes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutagênese , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Água/química , Zinco/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamas/química
14.
J Virol ; 80(10): 5050-8, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16641296

RESUMO

Norwalk virus (NV), a member of the Caliciviridae family, is the major cause of acute, epidemic, viral gastroenteritis. The NV genome is a positive sense, single-stranded RNA that encodes three open reading frames (ORFs). The first ORF produces a polyprotein that is processed by the viral cysteine protease into six nonstructural proteins. We have determined the structure of the NV protease to 1.5 and 2.2 A from crystals grown in the absence or presence, respectively, of the protease inhibitor AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride]. The protease, which crystallizes as a stable dimer, exhibits a two-domain structure similar to those of other viral cysteine proteases with a catalytic triad composed of His 30, Glu 54, and Cys 139. The native structure of the protease reveals strong hydrogen bond interactions between His 30 and Glu 54, in the favorable syn configuration, indicating a role of Glu 54 during proteolysis. Mutation of this residue to Ala abolished the protease activity, in a fluorogenic peptide substrate assay, further substantiating the role of Glu 54 during proteolysis. These observations contrast with the suggestion, from a previous study of another norovirus protease, that this residue may not have a prominent role in proteolysis (K. Nakamura, Y. Someya, T. Kumasaka, G. Ueno, M. Yamamoto, T. Sato, N. Takeda, T. Miyamura, and N. Tanaka, J. Virol. 79:13685-13693, 2005). In the structure from crystals grown in the presence of AEBSF, Glu 54 undergoes a conformational change leading to disruption of the hydrogen bond interactions with His 30. Since AEBSF was not apparent in the electron density map, it is possible that these conformational changes are due to subtle changes in pH caused by its addition during crystallization.


Assuntos
Cisteína Endopeptidases/química , Vírus Norwalk/química , Vírus Norwalk/enzimologia , Proteínas Virais/química , Ligação Competitiva , Domínio Catalítico , Cromatografia em Gel , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Dimerização , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Sulfonas/química , Proteínas Virais/metabolismo
15.
J Virol ; 76(19): 9832-43, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12208961

RESUMO

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein sigma 3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in sigma 3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the sigma 3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant sigma 3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced sigma 3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in sigma3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant sigma 3 proteins revealed structural alterations in sigma 3 that correlate with the Y354H mutation. These results indicate that a single mutation in sigma 3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of sigma 3 cleavage during reovirus disassembly.


Assuntos
Proteínas do Capsídeo , Capsídeo/química , Proteínas de Ligação a RNA , Montagem de Vírus , Capsídeo/metabolismo , Endocitose , Endopeptidases/fisiologia , Cinética , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes/biossíntese , Relação Estrutura-Atividade , Vírion/química , Vírion/crescimento & desenvolvimento
16.
Proc Natl Acad Sci U S A ; 98(4): 1381-6, 2001 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-11171959

RESUMO

Understanding the structural organization of the genome is particularly relevant in segmented double-stranded RNA viruses, which exhibit endogenous transcription activity. These viruses are molecular machines capable of repeated cycles of transcription within the intact capsid. Rotavirus, a major cause of infantile gastroenteritis, is a prototypical segmented double-stranded RNA virus. From our three-dimensional structural analyses of rotavirus examined under various chemical conditions using electron cryomicroscopy, we show here that the viral genome exhibits a remarkable conformational flexibility by reversibly changing its packaging density. In the presence of ammonium ions at high pH, the genome condenses to a radius of approximately 180 A from approximately 220 A. Upon returning to physiological conditions, the genome re-expands and fully maintains its transcriptional properties. These studies provide further insights into the genome organization and suggest that the observed isometric and concentric nature of the condensation is due to strong interactions between the genome core and the transcription enzymes anchored to the capsid inner surface. The ability of the genome to condense beyond what is normally observed in the native virus indicates that the negative charges on the RNA in the native state may be only partially neutralized. Partial neutralization may be required to maintain appropriate interstrand spacing for templates to move around the enzyme complexes during transcription. Genome condensation was not observed either with increased cation concentrations at normal pH or at high pH without ammonium ions. This finding indicates that the observed genome condensation is a synergistic effect of hydroxyl and ammonium ions involving disruption of protein-RNA interactions that perhaps facilitate further charge neutralization and consequent reduction in the interstrand spacing.


Assuntos
Genoma Viral , RNA de Cadeia Dupla/ultraestrutura , RNA Viral/ultraestrutura , Rotavirus/genética , Animais , Linhagem Celular , Microscopia Crioeletrônica/métodos , Meios de Cultura , Concentração de Íons de Hidrogênio , Conformação de Ácido Nucleico , Compostos de Amônio Quaternário , RNA de Cadeia Dupla/química , RNA Viral/química , Rotavirus/ultraestrutura , Transcrição Genética
17.
J Virol ; 74(14): 6546-55, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10864668

RESUMO

Aquareovirus, a member of the family Reoviridae, is a large virus with multiple capsid layers surrounding a genome composed of 11 segments of double-stranded RNA. Biochemical studies have shown that treatment with the proteolytic agent trypsin significantly alters the infectivity of the virus. The most infectious stage of the virus is produced by a 5-min treatment with trypsin. However, prolonged trypsin treatment almost completely abolishes the infectivity. We have used three-dimensional electron cryomicroscopy to gain insight into the structural basis of protease-induced alterations in infectivity by examining the structural changes in the virion at various time intervals of trypsin treatment. Our data show that after 5 min of trypsinization, projection-like spikes made of VP7 (35 kDa), associated with the underlying trimeric subunits, are completely removed. Concurrent with the removal of VP7, conformational changes are observed in the trimeric subunit composed of putative VP5 (71 kDa). The removal of VP7 and the accompanied structural changes may expose regions in the putative VP5 important for cell entry processes. Prolonged trypsinization not only entirely removes the outer capsid layer, producing the poorly infectious core particle, but also causes significant conformational changes in the turret protein. These changes result in shortening of the turret and narrowing of its central channel. The turret, as in orthoreoviruses, is likely to play a major role in the capping and translocation of mRNA during transcription, and the observed conformational flexibility in the turret protein may have implications in rendering the particle transcriptionally active or inactive.


Assuntos
Antígenos Virais , Proteínas do Capsídeo , Capsídeo/química , Reoviridae/ultraestrutura , Tripsina/farmacologia , Vírion/ultraestrutura , Animais , Bass/virologia , Microscopia Crioeletrônica , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Reoviridae/química , Reoviridae/patogenicidade , Vírion/química , Vírion/patogenicidade
18.
J Infect Dis ; 181 Suppl 2: S317-21, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10804144

RESUMO

Norwalk virus is the major cause of epidemic viral gastroenteritis in humans. Attempts to grow this human virus in laboratory cell lines have been unsuccessful; however, the Norwalk virus capsid protein, when expressed in insect cells infected with a recombinant baculovirus, spontaneously assembles into virus-like particles. The x-ray crystallographic structure of these recombinant Norwalk particles has been determined to 3.4 A, using a 22-A electron cryomicroscopy structure as a phasing model. The recombinant capsids, 380 A in diameter, exhibit a T=3 icosahedral symmetry. The capsid is formed by 90 dimers of the capsid protein, each of which forms an arch-like capsomere. The capsid protein has two distinct domains-a shell (S) and a protruding (P) domain-that are connected by a flexible hinge. Although the S domain has a classical beta-sandwich fold, the structure of the P domain is unlike any other viral protein. One of the subdomains in the P domain formed by the most variable part of the sequence is located at the exterior of the capsid.


Assuntos
Capsídeo/química , Vírus Norwalk/química , Cristalografia por Raios X , Proteínas Recombinantes/química
19.
Methods Mol Med ; 34: 9-31, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-21318853

RESUMO

Rotavirus (RV), a double-stranded (ds)RNA virus in the family Reoviridae, is a complex, relatively large (diameter, including spikes = 1000 Å), nonenveloped icosahedral virus. Once RV was recognized as a major human pathogen, it was extensively studied using modern molecular genetic and biological techniques, as discussed elsewhere in this book. These studies provided basic information about gene-coding assignments, protein processing, genome expression and replication, viral morphogenesis, and pathogenesis (1). In addition, molecular epidemiological studies, coupled with the characterization of neutralizing monoclonal antibodies (MAbs) and sequencing of the genes that encode the neutralizing antigens, provided an understanding at the molecular level of the antigenic and genetic variability of the RVs.

20.
J Struct Biol ; 116(1): 209-15, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8742745

RESUMO

The three-dimensional reconstruction of icosahedral viruses from electron micrograph images has become an important tool for understanding virus structure, function, and pathogenesis. We have developed an integrated suite of software programs to automate most of the operations involved in producing these reconstructions from EM images. Our package combines the analytical capabilities of preexisting algorithms together with approaches we have developed to produce an interactive working environment which enhances the efficiency and usefulness of this approach to the structural analysis of icosahedral viruses.


Assuntos
Simulação por Computador , Microscopia Eletrônica , Modelos Estruturais , Software , Vírus/ultraestrutura , Algoritmos , Automação , Gráficos por Computador , Análise de Fourier , Rotavirus/ultraestrutura
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