RESUMEN
Fifteen years after the discovery of hepatitis C virus (HCV) in 1989, much remains to be learnt about the cell biology of this virus. Using the serum from a patient containing HCV RNA in high titer as a source, a Singapore strain of genotype 1b was recovered and characterized. This full-length HCV genome was then constructed into a tetracycline-inducible vector using the pSTAR plasmid. Transfection of hepatoma cell lines with this HCV genome under tetracycline induction indicated that chemokines (RANTES and monocyte chemoattractant protein-1) were upregulated, possibly contributing to the induction of immune responses. Using the yeast two-hybrid system to discover protein-protein interactions, nonstructural region NS3 was found to interact with itself, forming a dimer that increased helicase activity but was not essential for its activity, thereby disqualifying it as a suitable target of drug actions. The significance of the interaction between core and NS5A is unclear, and the cleavage of NS5A is related to the development of apoptosis. However, the interaction of p68 and NS56B appears to be important because the knockdown of p68 reduced the viral replication. Finally, a new cell model using chimeric CD81 linked to the cytoplasmic domain of either a low-density lipoprotein receptor or a transferrin receptor led to productive infection of HCV that had been recovered from infected serum. These studies allow us to examine the pathogenesis of HCV infection in more detail.
Asunto(s)
Hepacivirus/fisiología , Hepatitis C/virología , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , ARN Helicasas DEAD-box , Genoma Viral , Hepacivirus/metabolismo , Hepatocitos/metabolismo , Plásmidos , Unión Proteica , Proteínas Quinasas/metabolismo , ARN Helicasas/metabolismo , Receptores de Lipoproteína/metabolismo , Receptores de Transferrina/metabolismo , Receptores Virales , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 28 , Transfección/métodos , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Proteínas de la Nucleocápside/inmunología , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Adolescente , Adulto , Western Blotting/métodos , Proteínas de la Nucleocápside de Coronavirus , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/genética , Valor Predictivo de las Pruebas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genéticaRESUMEN
Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these "accessory" viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de la Membrana/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas Virales/metabolismo , Animales , Células COS , Caspasa 3 , Línea Celular , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas de la Membrana/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Células Vero , Proteínas de la Matriz Viral , Proteínas Virales/genéticaRESUMEN
NS3, a non-structural protein of the HCV (hepatitis C virus), contains a protease and a helicase domain and plays essential roles in the processing of the viral polyprotein, viral RNA replication and translation. LMP7 (low-molecular-mass protein 7), a component of the immunoproteasome, was identified as an NS3-binding protein from yeast two-hybrid screens, and this interaction was confirmed by in vitro binding and co-immunoprecipitation analysis. The minimal domain of interaction was defined to be between the pro-sequence region of LMP7 (amino acids 1-40) and the protease domain of NS3. To elucidate the biological importance of this interaction, we studied the effect of this interaction on NS3 protease activity and on LMP7 immunoproteasome activity. Recombinant LMP7 did not have any effect on NS3 protease activity in vitro. The peptidase activities of LMP7 immunoproteasomes, however, were markedly reduced when tested in a stable cell line containing a HCV subgenomic replicon. The down-regulation of proteasome peptidase activities could interfere with the processing of viral antigens for presentation by MHC class I molecules, and may thus protect HCV from host immune surveillance mechanisms to allow persistent infection by the virus.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Biblioteca de Genes , Células HeLa/enzimología , Humanos , Inmunoprecipitación/métodos , Complejos Multienzimáticos/inmunología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasoma , Mapeo de Interacción de Proteínas/métodos , Bazo/química , Bazo/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/inmunología , Levaduras/genéticaRESUMEN
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxxphi and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.
Asunto(s)
Transporte Biológico , Endocitosis , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Humanos , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Conejos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Transfección , Células Vero , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoV-infected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Señales de Clasificación de Proteína , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Especificidad de la Especie , Transfección , Células Vero , Proteínas de la Matriz Viral , Proteínas Virales/químicaRESUMEN
Chronic infection by HCV is closely correlated with liver diseases such as cirrhosis, steatosis, and hepatocellular carcinoma. To understand how long-term interaction between HCV and the host leads to pathogenesis, we identified cellular proteins that interact with NS5A and NS5B using a biochemical approach. Stable cell lines that express flag-NS5A or flag-NS5B under tetracycline induction were generated. The induced flag-tagged proteins were immunoprecipitated (IP'd) and associated proteins separated on 2D gels. Protein spots that specifically co-IP'd with NS5A or NS5B were identified by mass spectrometry. HSP27 was identified as a protein that specifically co-IP'd with NS5A but not with NS5B. The N-terminal regions of NS5A (a.a. 1-181) and HSP27 (a.a. 1-122) were defined to be the domains that interact with each other. HSP27 is generally distributed in the cytoplasm. When heat shocked, HSP27 is concentrated in the ER where NS5A is co-localized.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS , Línea Celular , Chlorocebus aethiops , Electroforesis en Gel Bidimensional , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Proteínas de Choque Térmico HSP27 , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Pruebas de Precipitina , Proteómica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transfección , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Chronic infection by hepatitis C virus (HCV) can lead to severe hepatitis and cirrhosis and is closely associated with hepatocellular carcinoma. The replication cycle of HCV is poorly understood but is likely to involve interaction with host factors. In this report, we show that NS5B, the HCV RNA-dependent RNA polymerase (RdRp), interacts with a human RNA helicase, p68. Transient expression of NS5B alone, as well as the stable expression of all the nonstructural proteins in a HCV replicon-bearing cell line (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113), causes the redistribution of endogenous p68 from the nucleus to the cytoplasm. Deletion of the C-terminal two-thirds of NS5B (NS5BDeltaC) dramatically reduces its coimmunoprecipitation (co-IP) with endogenous p68, while the deletion of the N-terminal region (NS5BDeltaN1 and NS5BDeltaN2) does not affect its interaction with p68. In consistency with the co-IP results, NS5BDeltaC does not cause the relocalization of p68 whereas NS5BDeltaN1 does. With a replicon cell line, we were not able to detect a change in positive- and negative-strand synthesis when p68 levels were reduced using small interfering RNA (siRNA). In cells transiently transfected with a full-length HCV construct, however, the depletion (using specific p68 siRNA) of endogenous p68 correlated with a reduction in the transcription of negative-strand from positive-strand HCV RNA. Overexpression of NS5B and NS5BDeltaN1, but not that of NS5BDeltaC, causes a reduction in the negative-strand synthesis, indicating that overexpressed NS5B and NS5BDeltaN1 sequesters p68 from the replication complexes (thus reducing their replication activity levels). Identification of p68 as a cellular factor involved in HCV replication, at least for cells transiently transfected with a HCV expression construct, is a step towards understanding HCV replication.
Asunto(s)
Hepacivirus/fisiología , Proteínas Quinasas/fisiología , ARN Helicasas/fisiología , ARN Viral/biosíntesis , Proteínas no Estructurales Virales/fisiología , Replicación Viral , Núcleo Celular/enzimología , Citoplasma/enzimología , ARN Helicasas DEAD-box , Células HeLa , Hepacivirus/genética , Humanos , Transporte de Proteínas , ReplicónRESUMEN
An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/inmunología , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Valor Predictivo de las Pruebas , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.
Asunto(s)
Biomarcadores , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Antígenos Virales/genética , Antígenos Virales/inmunología , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Plásmidos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
In this study, we used HIV-1 as a model to demonstrate a novel approach for receptor-independent cell entry of virus. The heavy chain of an anti-HIV-1 gp120 antibody was engineered with endocytotic and transmembrane motifs from either the cation-independent mannose 6-phosphate receptor or the low-density lipoprotein receptor. Flow cytometry and immunofluorescence studies showed that the chimeric antibodies were expressed on the cell surface and can undergo rapid internalization. Furthermore, one of the chimeric antibodies was able to bind and internalize HIV-1. Using a luciferase reporter HIV-1, we further showed that internalized viruses could undergo replication. Therefore, we have demonstrated a proof-of-principle of a novel method that can be used to internalize virus into cells, without prior knowledge of the cellular receptor for the virus. We propose that this approach would be particularly useful for studying viruses whose cellular receptor(s) is not known.
Asunto(s)
Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/patogenicidad , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Endocitosis , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genes de Inmunoglobulinas , Genes Reporteros , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana , Datos de Secuencia MolecularRESUMEN
Although CD81 has been shown to bind HCV E2 protein, its role as a receptor for HCV remains controversial. In this study, we constructed two CD81 chimeras by linking the cytoplasmic domains of recycling surface receptors, low-density lipoprotein receptor (LDLR), and transferrin receptor (TfR), respectively, to CD81 and compared their internalization properties to wild-type CD81. Binding experiments with anti-hCD81 antibody showed that cell-surface CD81 chimeric receptors were internalized much more efficiently than wild-type CD81. In addition, CD81 chimeras, but not wild-type CD81, could internalize recombinant E2 protein and E2-enveloped viral particles from the serum of HCV-infected patients into Huh7 liver cells. The latter resulted in persistent positive-strand viral RNA and accumulation of replication intermediates, negative-strand viral RNA, in the infected cells, suggesting that the internalized viruses have undergone replication. Therefore, it appeared that CD81, possibly in association with a liver-specific endocytotic protein(s), represents one of the pathways by which HCV can infect hepatocytes.
Asunto(s)
Antígenos CD/metabolismo , Endocitosis , Hepacivirus/fisiología , Proteínas de la Membrana/metabolismo , Receptores Virales/fisiología , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 28 , Virión/fisiología , Replicación ViralRESUMEN
We had previously reported the cloning of the complete genome of an isolate of hepatitis C virus (HCV), HCV-S1, of genotype 1b. We have constructed a full-length complementary DNA (cDNA) clone of HCV-S1 using nine overlapping cDNA clones that encompassed its entire genome. HCV core, E1, E2, NS-3, -4B, -5A, and -5B proteins were detected in 293T cells by immunoblot analyses when expression of the full-length HCV-S1 was driven under a CMV promoter. Expression of full-length HCV-S1 led to induction of the CC chemokines RANTES and MCP-1 at both the mRNA and the protein levels in HeLa, Huh7, and HepG2 cells. Reporter gene assays showed that a minimal MCP-1 promoter construct containing 128 nucleotides upstream of its translational start site was sufficient for optimal HCV-mediated activation. HCV induced AP-1 binding activities to this region, as determined from electrophoretic mobility shift assays and supershifts with anti-AP-1 antibodies. Transfection of full-length HCV-S1 up-regulated both AP-1 binding activities as well as c-jun transcripts. A minimal promoter construct containing 181 nucleotides upstream of the RANTES translational start site was sufficient for maximal HCV-mediated induction. Gel mobility shift and supershift assays showed that HCV induced NF-kappaB and other unknown binding activities to the A/B-site within this region. In HeLa cells, HCV core and NS5A could separately augment promoter activities of both MCP-1 and RANTES. In Huh7 cells, only NS5A produced a similar effect, while rather surprisingly, HCV core induced a dramatic reduction in promoter activities of these two genes. This study provides the first direct evidence for the induction of CC chemokines in HCV infection and draws attention to their roles in affecting the progress and outcome of HCV-associated liver diseases.