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1.
Clin Chem Lab Med ; 61(8): 1511-1517, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-36799248

RESUMO

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Sensibilidade e Especificidade , Teste para COVID-19 , Imunoensaio
2.
J Clin Microbiol ; 56(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29743308

RESUMO

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.


Assuntos
Anticorpos Antiprotozoários/sangue , Babesia microti/isolamento & purificação , Babesiose/diagnóstico , Imunoensaio/normas , Parasitemia/diagnóstico , Animais , Anticorpos Antiprotozoários/imunologia , Babesia microti/genética , Babesia microti/imunologia , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo/normas , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Macaca , Programas de Rastreamento , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Soroconversão , Reação Transfusional/prevenção & controle
3.
Biotechniques ; 73(3): 136-141, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36004516

RESUMO

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/diagnóstico , COVID-19/genética , Teste para COVID-19 , Testes Diagnósticos de Rotina , Humanos , Mutação , Nucleocapsídeo/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade
4.
BMC Biochem ; 12: 1, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21205312

RESUMO

BACKGROUND: Like all tripartite motif (TRIM) proteins, the retroviral restriction factor TRIM5α consists of RING, B-box 2 and coiled-coil domains, with a C-terminal B30.2(SPRY) domain. Although structures have been determined for some individual TRIM domains, the structure of an intact TRIM protein is unknown. RESULTS: Here, we express and characterize a protease-resistant 29-kD core fragment containing the B-box 2, coiled coil and adjacent linker (L2) region of TRIM5α. This BCCL2 protein formed dimers and higher-order oligomers in solution. Approximately 40% of the BCCL2 secondary structure consisted of alpha helices. Partial loss of alpha-helical content and dissociation of dimers occurred at 42°C, with the residual alpha helices remaining stable up to 80°C. CONCLUSIONS: These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.


Assuntos
Estrutura Secundária de Proteína/genética , Proteínas/química , Proteínas/genética , Animais , Dimerização , Escherichia coli , Macaca mulatta , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Ubiquitina-Proteína Ligases , Zinco/metabolismo
5.
J Virol ; 83(21): 10951-62, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19656870

RESUMO

The binding of cyclophilin A (CypA) to the human immunodeficiency virus type 1 (HIV-1) capsid protein (CA protein) is required soon after virus entry into natural target cells. In Jurkat T lymphocytes, disrupting CypA-CA interaction either by cyclosporine (Cs) treatment or by alteration (e.g., P90A) of the CA inhibits HIV-1 infection. In HeLa cells, however, treatment with Cs or Cs analogues minimally inhibits the early phase of HIV-1 infection but selects for a Cs-dependent virus with a change (A92E) in CA. To understand these phenomena, we examined the effects of the P90A and A92E changes in the HIV-1 CA protein on the stability of capsid complexes assembled in vitro and on capsid disassembly in the cytosol of virus-exposed target cells. The A92E change impaired CA-CA interactions in vitro and decreased the amount of particulate capsids in the cytosol of HeLa target cells. Reducing the binding of CypA to the A92E mutant capsid, either by Cs treatment or by an additional P90A change in the CA protein, increased the amount of particulate capsids and viral infectivity in HeLa cells. In contrast, reduction of the binding of CypA to HIV-1 capsids in Jurkat T lymphocytes resulted in a decrease in the amount of particulate capsids and infectivity. Thus, depending on the capsid and the target cell, CypA-CA binding either stabilized or destabilized the capsid, indicating that CypA modulates HIV-1 capsid disassembly. In both cell types examined, decreased stability of the capsid was associated with a decrease in the efficiency of HIV-1 infection.


Assuntos
Proteínas do Capsídeo/metabolismo , Ciclofilina A/metabolismo , HIV-1/metabolismo , Substituição de Aminoácidos , Proteínas do Capsídeo/genética , Ciclofilina A/genética , HIV-1/genética , HIV-1/patogenicidade , HIV-1/ultraestrutura , Células HeLa , Humanos , Células Jurkat , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
6.
J Virol ; 82(23): 11669-81, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18799572

RESUMO

The tripartite motif (TRIM) protein, TRIM5alpha, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5alpha promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5alpha protein (TRIM5(Rh)-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5(Rh)-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5(Rh)-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5(Rh)-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5(Rh)-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Proteínas/química , Proteínas Recombinantes de Fusão/química , Animais , Capsídeo/metabolismo , Dicroísmo Circular , Proteínas de Ligação a DNA/fisiologia , Dimerização , HIV-1/metabolismo , Humanos , Proteínas Nucleares/fisiologia , Proteínas/fisiologia , Coelhos , Ribonucleoproteínas , Spodoptera , Ubiquitina-Proteína Ligases/metabolismo
7.
J Virol ; 82(23): 11682-94, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18799573

RESUMO

The rhesus monkey intrinsic immunity factor TRIM5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5alpha(rh) protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5alpha proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5alpha proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5alpha proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.


Assuntos
Proteínas de Ligação a DNA/química , HIV-1/fisiologia , Proteínas Nucleares/química , Proteínas/química , Proteínas Recombinantes de Fusão/química , Replicação Viral , Capsídeo/química , Capsídeo/metabolismo , Proteínas de Ligação a DNA/fisiologia , Dimerização , Células HeLa , Humanos , Proteínas Nucleares/fisiologia , Nucleocapsídeo/metabolismo , Estrutura Terciária de Proteína , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Ribonucleoproteínas , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
BMC Mol Biol ; 8: 4, 2007 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-17241458

RESUMO

BACKGROUND: Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function. RESULTS: Our data revealed that expression of full-length NS2 was sufficient for the formation of inclusion bodies (IBs) that were morphologically similar to the VIBs formed during BTV infection. By using either, individual BTV proteins or infectious virions, we found that while the VP3 of the inner capsid (termed as "core") that surrounds the transcription complex was closely associated with both NS2 IBs and BTV VIBs, the surface core protein VP7 co-localized with NS2 IBs only in the presence of VP3. In contrast to the inner core proteins, the outer capsid protein VP2 was not associated with either IBs or VIBs. Like the core proteins, newly synthesized BTV RNAs also accumulated in VIBs. Unlike full-length NS2, neither the amino-, nor carboxyl-terminal fragments formed complete IB structures and each appeared to interfere in overall virus replication when similarly expressed. CONCLUSION: Together, these data demonstrate that NS2 is sufficient and necessary for IB formation and a key player in virus replication and core assembly. Perturbation of NS2 IB formation resulted in reduced virus synthesis and both the N terminal (NS2-1) and C terminal (NS2-2) fragments act as dominant negative mutants of NS2 function.


Assuntos
Vírus Bluetongue/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas não Estruturais Virais/fisiologia , Replicação Viral , Animais , Vírus Bluetongue/genética , Linhagem Celular , Regulação Viral da Expressão Gênica , Corpos de Inclusão/virologia , Mamíferos , Proteínas de Ligação a RNA/genética , Transfecção , Proteínas não Estruturais Virais/genética
9.
PLoS One ; 9(6): e95032, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24893007

RESUMO

Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Lisossomos/metabolismo , Proteólise/efeitos dos fármacos , Aldeídos/farmacologia , Animais , Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina C/antagonistas & inibidores , Catepsina C/metabolismo , Dipeptídeos/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Leupeptinas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Necrose , Peptídeos/farmacologia , Inibidores de Proteassoma/farmacologia
10.
Virology ; 369(2): 400-10, 2007 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-17920096

RESUMO

The restriction factors, TRIM5alpha in most primates and TRIMCyp in owl monkeys, block infection of various retroviruses soon after virus entry into the host cell. Rhesus monkey TRIM5alpha (TRIM5alpha rh) inhibits human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV) more potently than human TRIM5alpha (TRIM5alpha hu). TRIMCyp restricts infection of HIV-1, simian immunodeficiency virus of African green monkeys (SIV agm) and FIV. Early after infection, TRIMCyp, like TRIM5alpha rh and TRIM5alpha hu, decreased the amount of particulate viral capsid in the cytosol of infected cells. The requirements for the TRIMCyp and TRIM5alpha domains in restricting different retroviruses were investigated. Potent restriction of FIV by TRIMCyp occurred in the complete absence of RING and B-box 2 domains; by contrast, efficient FIV restriction by TRIM5alpha rh required these domains. Variable region 1 of the TRIM5alpha rh B30.2 domain contributed to the potency of HIV-1, FIV and equine infectious anemia virus restriction. Thus, although differences exist in the requirements of TRIMCyp and TRIM5alpha for RING/B-box 2 domains, both restriction factors exhibit mechanistic similarities.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas/fisiologia , Infecções por Retroviridae/fisiopatologia , Animais , Fatores de Restrição Antivirais , Aotidae , Capsídeo/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Gatos , Linhagem Celular , HIV-1/genética , HIV-1/fisiologia , Humanos , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/fisiologia , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Infecções por Retroviridae/virologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
11.
J Virol ; 81(19): 10362-78, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17626085

RESUMO

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , HIV-1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Retroviridae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Fatores de Restrição Antivirais , Arginina/química , Arginina/genética , Capsídeo/metabolismo , Proteínas de Transporte/genética , Humanos , Macaca mulatta , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Estrutura Terciária de Proteína , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
12.
J Virol ; 79(17): 11487-95, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16103199

RESUMO

The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.


Assuntos
Vírus Bluetongue/fisiologia , Proteínas do Core Viral/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Corpos de Inclusão Viral/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus
13.
J Virol ; 77(21): 11347-56, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14557620

RESUMO

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.


Assuntos
Vírus Bluetongue/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Células Cultivadas , Dados de Sequência Molecular , Mutação , Nucleopoliedrovírus/genética , RNA Helicases/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Spodoptera , Relação Estrutura-Atividade
14.
Virology ; 324(2): 387-99, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15207624

RESUMO

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.


Assuntos
Vírus Bluetongue/fisiologia , Proteínas do Core Viral/biossíntese , Vírus Bluetongue/ultraestrutura , Proteínas do Capsídeo/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Mutação , Proteínas Recombinantes/biossíntese , Sequências Repetidas Terminais , Transcrição Gênica , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Montagem de Vírus
15.
J Virol ; 78(15): 8059-67, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15254177

RESUMO

Bluetongue virus is a large and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA genome. X-ray crystallographic analysis of the particles without the outer capsid layer has provided atomic structural details of VP3 and VP7, which form the inner two layers. However, limited structural information is available on the other five proteins in the virion-two of which are important for receptor recognition, hemagglutination, and membrane interaction-are in the outer layer, and the others, important for endogenous transcriptase activity are internal. Here we report the electron cryomicroscopy (cryo-EM) reconstruction of the mature particle, which shows that the outer layer has a unique non-T = 13 icosahedral organization consisting of two distinct triskelion and globular motifs interacting extensively with the underlying T = 13 layer. Comparative cryo-EM analysis of the recombinant corelike particles has shown that VP1 (viral polymerase) and VP4 (capping enzyme) together form a flower-shaped structure attached to the underside of VP3, directly beneath the fivefold axis. The structural data have been substantiated by biochemical studies demonstrating the interactions between the individual outer and inner capsid proteins.


Assuntos
Vírus Bluetongue/química , Capsídeo/química , Proteínas do Capsídeo/química , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Transcrição Gênica , Proteínas do Core Viral/química
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