- Email: [email protected]
HIV co-infection
332A 901
AASLD HCV CORE PROTEIN SUBCELLULAR LOCALIZATION: ANALYSIS BY CONFOCAL AND ELECTRON MICROSCOPY AND CORRELATION WITH THE CELL CYCLE. wz. T I&&& HalDer$, Y Matsuurai. T Mivunurag. C Brechod. *Liver cancer and molecular virology, InsermU370, Paris, France. SDepartment of Virology II, National Institute of Health, Tokyo, Japan. $ Laboratoire de Biologie et Ultrastructure du Noyau, CNRS UPR272, ViIlejuif, France. Immunofluorescence and confocal microscopy were performed to analyze the subcellular distribution of the core protein in CHO and HepG2 cells stably expressing core and El proteins. The core protein revealed by immunostaining is cytoplasmic with a perinuclear pattern localized in globular structures. In few cells these structures were found also in nucleus. The nuclear localization was confirmed by confocal analysis. To investigate if this nuclear localization could be cell-cycle regulated, cells were blocked in late Gl phase by overnight treatment with 3mM hydroxyurea and then released in normal medium. The synchronization did not affect the distribution pattern of the core protein. The particle distribution found in the cytoplasm was further analyzed by electron microscopy. Comparative morphological analysis of cells transfected by the vector only (negative control) and the core + El construct showed in the latter the presence of lipidic (triglycerld) vesicles of about lOOnm, not present in the control cells. Immunostaining with an anti-core antibody revealed aconcentration of the signal on the exterior surface of the vesicles. Addition of oleic acid in. Hep G2 cells medium increased the size of these triglycerides ccmteinmg vesicles where HCV core could be localized. Further studies are in progress to analyze the localization of the El protein in these cells. Conclusions : our analysis : 1) shows both nuclear and cytoplasmic localisation of HCV core 2) does not support cell cycle regulation of HCV core localisation 3) suggests that HCV core expression is associated to modification of cellular lipid metabolism 4) is consistent with the association of HCV core in the cell to Triglycerides containing vesicles.
903 SINGLE-STRAND
CONFORMATION
POLYMORPHISM
REGION. BR Lareu. WD Reed and NR Swanson. University Dept. Medicine and Dept. Gastroenterology, QEIIMC, Nedlands, Westem Auahlia 6009. ANALYSIS
OF THE HCV
HYPERVARIAB=
Rapid mutation of the hyper-variable region (HVR) of the putative HCV envelope glycoprotein E2 may be a major mechahm involved in evasion of the host immune system leading to a state of chronic viral infection. The aim of the present study was to invest+ the tslmique of single-straud conformation polymorphism (SSCP) asa meausof rapid smening of HVR diversity in chronic hepatitis C (CHC). SSCP is a rapid and simple technique for detect& sequm &f&axes between DNA @meats. A nested-asymmuk PCR assay was developed to amplify part of the putative E2 gene conthiag the HVR, giving single+ stranded DNA fhgments which were then analysed by SSCP on nondenaturhg polyacryhide gels. CHC patients treated with IFN were assessedprior, during and post-therapy. Primers immed&Iy &king the HVR gave rise to a high proportion of tklsenegative results due to moderate sequencevariation. Alternate primers were designed for more cowed but distal Sanking regions. This overcame the problem of false-negativity hut resulted in a large 576bp PCR product. Asymmetric-PCR prevented conksion when analyzing multiple bands correspondiogtoseveralHCVvariants(iclbandinsteadof2pa variant). Excision of baads from dried SSCP gels followed by a single inner PCR reaction enabled linther analysis of individual variant complexity by direct sequencing. Our SSCP assay contkmed the presenceof single to multiple E2 variant virus part&s per serum sample analyzed.Greatest HVR diversity and sequencecomplexity was found in those patients who did not respond to IPN. We have concluded that SSCP may be suuxs&lly used to analyze the PCR products in most CHC patients..Our results show a trend between a low number of E2 variants prior to IPN therapy and a good responseto IFN.
ABSTRACTS
HEPATOLOGY
902 MATURATION AND BIOLOGICAL HEPATITIS C VIRUS CORE PROTEIN.
October
IMPORTANCE
1995
OF
K.l. 2, T. Wakital, K. T-Koharal, M.Ichikawa3 T. K@it&, A.Haseaawd, 1The Tokyo Metro. Institute’of Medical -M Koharal. Science, 23rd Dept. of Internal Medicine, Kyoto Prefectural Univ. of Medicine, 3The Tokyo Metro. Institute for Neuroscience, 41nternational Reagents Co., 5Corporate Research and Development Lab., Tonen Co. Hepatitis C virus (HCV) core protein consists viral particle and is considered to have other biological functions. It may play an important role in the pathogenesis of chronic HCV infection causing hepatocellular carcinoma in many cases. Native core protein was isolated from patient sera with chronic HCV infection. Recombinant vaccinia virus (RVV) expressing entire structural proteins (core, El, and E2) and NS2 protein was infected to RK13 and HPB-Ma cells. Stable transformants of structural proteins (core, El and E2) were also established with CHO cells. ELISA and western blotting assays determined the amounts and the sizes of the different forms of core proteins. Confocal laser scanning microscope was used to find the precise subcellular localization of the expressed proteins. Core proteins were detected in 80-90% of the serum samples from chronic hepatitis C patients by ELISA. High titer sera were selected for the further analysis. MW of native HCV core protein isolated from the patient sera was 2lkDa, and it appeared at the same size as expressed form of core protein from the RVV infected cells. The maturation of putative core protein from precursor polyprotein was also characterized both in the RVV infected cells and transformed CHO cell lines with different levels of protein expression. Confocal microscopic analysis revealed that the naturally processed core protein distributed both in the cytoplasm and nucleus. The sizes of cytoplasmic and nuclear core proteins were same 21 kDa. Phospholyration of the core protein was also evaluated. Furthermore, HCV stable transformant of CHO cells exhibited lower growth rate compared with non transformed cells. In conclusion, both native and expressed HCV core protein showed similar character in this study. Processed core protein was 21kDa and located both in the cytoplasm and nucleus. Expression of HCV core protein may regulate the cell growth by unknown mechanisms. Further analysis will determine the specific biological functions of core protein and its roles in the pathogenesis of chronic HCV infection.
904 INCREASED VARIABILITY IN THE HCV RNA HYPERVARIABLE REGION AMONG PATIENTS WITH HCV/HIV CO-INFECTION. KE Sherman*. C Andreatta, J O’Brien, A Gutierrez, R Harris .University of Cincinnati Medical Center, Dept. of Medicine and Pathology, Cincinnati, OH* and Fitzsimons Army Medical Center, Aurora, CO Patients co-infected with the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were studied with regard to nucleotide sequence variability in the E2/NSl first hypervariable regton of the HCV genome. The nucleotide variability within individual patients was compared to patients only infected with HCV. The proportion of predicted synonymous and non-synonomous amino acid changes, and the relationship to putative high antigenicity sites was evaluated in the hypervariable envelope domain. Ninety-one clones from 10 patients with HCV/HIV co-infection were sequenced. The control HCV group included 53 clones from 7 patients. Sequence analysis encompassed the region coding for amino acids 384-414. RESULTS: Cumulative proportional comparison at each amino acid site revealed increased variability in HCV RNA from patients with HCV/HIV co-infection vs. HCV alone. The greatest variability was seen at aa386, 389, 397, 400, 402, 405, 407, 413,and 414, with greater than 10% clonal variation at these sites. Jameson-Wolf plots were used to predict putative high antigenicity domains. Nonsynonomous clonal variation resulted in alteration of putative antigenic sites within the hypervariable region. All clones had at least one high probability site. Clones with unique predicted antigenic domains were observed more frequently in HIV/HCV co-infected patients, consistent with increased sequence variability. CONCLUSION These data suggest an accumulation of envelope variants in the HCV/HIV co-infected patients, which could be related to ineffective viral clearance, and may help explain prior reports of interferon resistance in this patient group.
AASLD HCV CORE PROTEIN SUBCELLULAR LOCALIZATION: ANALYSIS BY CONFOCAL AND ELECTRON MICROSCOPY AND CORRELATION WITH THE CELL CYCLE. wz. T I&&& HalDer$, Y Matsuurai. T Mivunurag. C Brechod. *Liver cancer and molecular virology, InsermU370, Paris, France. SDepartment of Virology II, National Institute of Health, Tokyo, Japan. $ Laboratoire de Biologie et Ultrastructure du Noyau, CNRS UPR272, ViIlejuif, France. Immunofluorescence and confocal microscopy were performed to analyze the subcellular distribution of the core protein in CHO and HepG2 cells stably expressing core and El proteins. The core protein revealed by immunostaining is cytoplasmic with a perinuclear pattern localized in globular structures. In few cells these structures were found also in nucleus. The nuclear localization was confirmed by confocal analysis. To investigate if this nuclear localization could be cell-cycle regulated, cells were blocked in late Gl phase by overnight treatment with 3mM hydroxyurea and then released in normal medium. The synchronization did not affect the distribution pattern of the core protein. The particle distribution found in the cytoplasm was further analyzed by electron microscopy. Comparative morphological analysis of cells transfected by the vector only (negative control) and the core + El construct showed in the latter the presence of lipidic (triglycerld) vesicles of about lOOnm, not present in the control cells. Immunostaining with an anti-core antibody revealed aconcentration of the signal on the exterior surface of the vesicles. Addition of oleic acid in. Hep G2 cells medium increased the size of these triglycerides ccmteinmg vesicles where HCV core could be localized. Further studies are in progress to analyze the localization of the El protein in these cells. Conclusions : our analysis : 1) shows both nuclear and cytoplasmic localisation of HCV core 2) does not support cell cycle regulation of HCV core localisation 3) suggests that HCV core expression is associated to modification of cellular lipid metabolism 4) is consistent with the association of HCV core in the cell to Triglycerides containing vesicles.
903 SINGLE-STRAND
CONFORMATION
POLYMORPHISM
REGION. BR Lareu. WD Reed and NR Swanson. University Dept. Medicine and Dept. Gastroenterology, QEIIMC, Nedlands, Westem Auahlia 6009. ANALYSIS
OF THE HCV
HYPERVARIAB=
Rapid mutation of the hyper-variable region (HVR) of the putative HCV envelope glycoprotein E2 may be a major mechahm involved in evasion of the host immune system leading to a state of chronic viral infection. The aim of the present study was to invest+ the tslmique of single-straud conformation polymorphism (SSCP) asa meausof rapid smening of HVR diversity in chronic hepatitis C (CHC). SSCP is a rapid and simple technique for detect& sequm &f&axes between DNA @meats. A nested-asymmuk PCR assay was developed to amplify part of the putative E2 gene conthiag the HVR, giving single+ stranded DNA fhgments which were then analysed by SSCP on nondenaturhg polyacryhide gels. CHC patients treated with IFN were assessedprior, during and post-therapy. Primers immed&Iy &king the HVR gave rise to a high proportion of tklsenegative results due to moderate sequencevariation. Alternate primers were designed for more cowed but distal Sanking regions. This overcame the problem of false-negativity hut resulted in a large 576bp PCR product. Asymmetric-PCR prevented conksion when analyzing multiple bands correspondiogtoseveralHCVvariants(iclbandinsteadof2pa variant). Excision of baads from dried SSCP gels followed by a single inner PCR reaction enabled linther analysis of individual variant complexity by direct sequencing. Our SSCP assay contkmed the presenceof single to multiple E2 variant virus part&s per serum sample analyzed.Greatest HVR diversity and sequencecomplexity was found in those patients who did not respond to IPN. We have concluded that SSCP may be suuxs&lly used to analyze the PCR products in most CHC patients..Our results show a trend between a low number of E2 variants prior to IPN therapy and a good responseto IFN.
ABSTRACTS
HEPATOLOGY
902 MATURATION AND BIOLOGICAL HEPATITIS C VIRUS CORE PROTEIN.
October
IMPORTANCE
1995
OF
K.l. 2, T. Wakital, K. T-Koharal, M.Ichikawa3 T. K@it&, A.Haseaawd, 1The Tokyo Metro. Institute’of Medical -M Koharal. Science, 23rd Dept. of Internal Medicine, Kyoto Prefectural Univ. of Medicine, 3The Tokyo Metro. Institute for Neuroscience, 41nternational Reagents Co., 5Corporate Research and Development Lab., Tonen Co. Hepatitis C virus (HCV) core protein consists viral particle and is considered to have other biological functions. It may play an important role in the pathogenesis of chronic HCV infection causing hepatocellular carcinoma in many cases. Native core protein was isolated from patient sera with chronic HCV infection. Recombinant vaccinia virus (RVV) expressing entire structural proteins (core, El, and E2) and NS2 protein was infected to RK13 and HPB-Ma cells. Stable transformants of structural proteins (core, El and E2) were also established with CHO cells. ELISA and western blotting assays determined the amounts and the sizes of the different forms of core proteins. Confocal laser scanning microscope was used to find the precise subcellular localization of the expressed proteins. Core proteins were detected in 80-90% of the serum samples from chronic hepatitis C patients by ELISA. High titer sera were selected for the further analysis. MW of native HCV core protein isolated from the patient sera was 2lkDa, and it appeared at the same size as expressed form of core protein from the RVV infected cells. The maturation of putative core protein from precursor polyprotein was also characterized both in the RVV infected cells and transformed CHO cell lines with different levels of protein expression. Confocal microscopic analysis revealed that the naturally processed core protein distributed both in the cytoplasm and nucleus. The sizes of cytoplasmic and nuclear core proteins were same 21 kDa. Phospholyration of the core protein was also evaluated. Furthermore, HCV stable transformant of CHO cells exhibited lower growth rate compared with non transformed cells. In conclusion, both native and expressed HCV core protein showed similar character in this study. Processed core protein was 21kDa and located both in the cytoplasm and nucleus. Expression of HCV core protein may regulate the cell growth by unknown mechanisms. Further analysis will determine the specific biological functions of core protein and its roles in the pathogenesis of chronic HCV infection.
904 INCREASED VARIABILITY IN THE HCV RNA HYPERVARIABLE REGION AMONG PATIENTS WITH HCV/HIV CO-INFECTION. KE Sherman*. C Andreatta, J O’Brien, A Gutierrez, R Harris .University of Cincinnati Medical Center, Dept. of Medicine and Pathology, Cincinnati, OH* and Fitzsimons Army Medical Center, Aurora, CO Patients co-infected with the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were studied with regard to nucleotide sequence variability in the E2/NSl first hypervariable regton of the HCV genome. The nucleotide variability within individual patients was compared to patients only infected with HCV. The proportion of predicted synonymous and non-synonomous amino acid changes, and the relationship to putative high antigenicity sites was evaluated in the hypervariable envelope domain. Ninety-one clones from 10 patients with HCV/HIV co-infection were sequenced. The control HCV group included 53 clones from 7 patients. Sequence analysis encompassed the region coding for amino acids 384-414. RESULTS: Cumulative proportional comparison at each amino acid site revealed increased variability in HCV RNA from patients with HCV/HIV co-infection vs. HCV alone. The greatest variability was seen at aa386, 389, 397, 400, 402, 405, 407, 413,and 414, with greater than 10% clonal variation at these sites. Jameson-Wolf plots were used to predict putative high antigenicity domains. Nonsynonomous clonal variation resulted in alteration of putative antigenic sites within the hypervariable region. All clones had at least one high probability site. Clones with unique predicted antigenic domains were observed more frequently in HIV/HCV co-infected patients, consistent with increased sequence variability. CONCLUSION These data suggest an accumulation of envelope variants in the HCV/HIV co-infected patients, which could be related to ineffective viral clearance, and may help explain prior reports of interferon resistance in this patient group.