A wide range of NS3/4A protease catalytic efficiencies in HCV-infected individuals

The hepatitis C virus (HCV) NS3/4A protease acts as an antagonist of virus-induced interferon (IFN) regulatory factor (IRF)-3 activation and IFN-beta expression. The NS3/4A protease performs this function by cleaving Cardif and TRIF proteins to block retinoic-acid-inducible gen I (RIG-I) and toll-like receptor (TLR)-3 signaling, respectively. NS3/4A protease inhibition can prevent Cardif and/or TRIF inactivation during HCV infection, thereby maintaining the innate immune response. Thus, differences in NS3/4A protease catalytic efficiency could be related to viral pathogenicity. In this study, we analyzed the catalytic efficiency of the most abundant NS3/4A protease isolated from each of 12 individuals infected with HCV genotypes 1b, 1a, 3a, 4a or 4d. A diversity of NS3/4A protease catalytic efficiencies (up to a six-fold difference) was found in the analyzed samples. The genotype 1b NS3/4A proteases displayed the highest catalytic efficiencies. However, within this genotype up to three-fold differences were observed. Cross-genotypic interactions between the NS3 protease domain and the NS4A cofactor peptide were also investigated. Overall, catalytic efficiencies of NS3 proteases cross-interacting with NS4A cofactors from heterologous genotypes were as efficient as the homologous NS3/4A interactions. Of the 28 heterologous interactions tested, only 6 resulted in deleterious or nonfunctional enzymes. Nonfunctional interactions were not genotype-specific, suggesting that enhancement of NS3 catalytic efficiency by the NS4A cofactor depends on a few specific amino acid residues. Characterization of the proteolytic activities of individual NS3/4A proteases should provide clues for understanding HCV-host interactions, as well as assisting in the development of new classes of NS3/4A protease inhibitors.     

Subcellular localisation and sedimentation behaviour of antigen 60 from Mycobacterium bovis BCG

Preparation, composition and immunological properties of A60 of Mycobacterium bovis BCG were previously described (Cocito and Vanlinden 1986). The present study focused on the intracellular distribution of this antigen. Fractionation of mycobacterial homogenates by ultracentrifugation indicated that most of A60 was present within the cytoplasm. Some of the antigen was located within the cell wall, from which it was released by extraction with alkali. Submission of cytoplasm to high speed centrifugation caused A60 to cosediment with ribosomes; however, dissociation of ribosomes in low-Mg buffer did not alter the sedimentation pattern of A60. Labelled A60, after ultracentrifugation in sucrose density gradients without Mg2+, was distributed throughout the entire gradient: treatment of (125I)A60 with urea or detergents produced a peak of radioactivity located in the upper part of the gradient. It is concluded that A60 is represented by a heterogeneous family of molecules of increasing sizes: polymerization being enhanced by Mg2+ and reversibly prevented by urea. Some or all of the biological properties hitherto attributed to ribosomal particles may, in fact, be due to their contamination with cosedimented A60.     

Subcellular localisation and sedimentation behaviour of antigen 60 from Mycobacterium bovis BCG

Preparation, composition and immunological properties of A60 of Mycobacterium bovis BCG were previously described (Cocito and Vanlinden 1986). The present study focused on the intracellular distribution of this antigen. Fractionation of mycobacterial homogenates by ultracentrifugation indicated that most of A60 was present within the cytoplasm. Some of the antigen was located within the cell wall, from which it was released by extraction with alkali. Submission of cytoplasm to high speed centrifugation caused A60 to cosediment with ribosomes; however, dissociation of ribosomes in low-Mg buffer did not alter the sedimentation pattern of A60. Labelled A60, after ultracentrifugation in sucrose density gradients without Mg²⁺, was distributed throughout the entire gradient: treatment of (¹²⁵I)A60 with urea or detergents produced a peak of radioactivity located in the upper part of the gradient. It is concluded that A60 is represented by a heterogeneous family of molecules of increasing sizes: polymerization being enhanced by Mg²⁺ and reversibly prevented by urea. Some or all of the biological properties hitherto attributed to ribosomal particles may, in fact, be due to their contamination with cosedimented A60.Abbreviations CMN Group of Gram-positive microorganisms including the genera Corynebacterium, Mycobacterium and Nocardia - BCG Mycobacterium bovis, strain Calmette-Guérin - TMA Thermostable macromolecular antigen, a family of mycobacterial antigens to which A60 belongs - PPD Purified protein derivative, according to F. B. Seibert (cf. Daniel and Janicki 1978) - OT Old tuberculin, according to R. Koch (cf. Youmans 1979b) - NMT Buffer containing NH₄Cl, Mg acetate and Tris (see Materials) - PBS Phosphate-buffered saline (see Materials) - A60 Antigen 60 of BCG (according to the classification of Closs et al. 1980)     

HCV NS3 N末端部分氨基酸缺失对NS3/NS3与NS3/NS4A分子间相互作用的影响

目的旨在弄清ＮＳ３参与分子间相互作用的确切区段，为研究针对ＮＳ３的抗ＨＣＶ寡肽小分子药物的设计提供依据。方法参照ＨＣＶ中国河北株序列设计ＮＳ３引物，将其Ｎ末端的前１５个和前３０个氨基酸分别缺失掉。然后用酵母双杂交系统检测ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用强度在缺失前后的变化，从而判明ＮＳ３Ｎ末端氨基酸在分子间相互作用中的意义。核苷酸序列分析采用ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍ３７３Ａ型自动测序仪。结果ＮＳ３Ｎ末端氨基酸缺失前后，ＮＳ３／ＮＳ３分子间及ＮＳ３／ＮＳ４Ａ分子间相互作用的强度相差有显著性（Ｐ＜０．０１），但缺失１５个氨基酸和缺失３０个氨基酸对上述相互作用强度的影响差异无显著性（Ｐ＞０．０５）。结论ＮＳ３Ｎ末端的１～３０个氨基酸在ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用中有一定意义，其Ｎ末端前１５个氨基酸（ＡＰＩＴＡＹＳＱＱＴＲＧＬＬＧ）对于分子间相互作用更为关键。本研究结果将为抗ＮＳ３丝氨酸蛋白酶活性的寡肽抑制物的研究打下基础，并为抗ＨＣＶ的寡肽小分子药物的设计提供依据     

Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome.

In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation. We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?). EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses. The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25. The analysis clearly shows that all five breakpoints are distinct. Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients. Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease. The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints. This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development.     

IgA localisation in glomerular diseases.

The structural changes found on light and electron microscopy study of 25 renal biopsy specimens that showed significant glomerular IgA deposition on immunofluorescence were correlated with relevant clinical data. The morphology of a wide range of glomerulopathies seeen included mesangial proliferative (60%), membranous (12%), rapidly progressive proliferative (8%), mesangio-capillary (8%), and no light microscope change (8%). Four of the 15 cases (60%) of mesangial proliferative glomerulonephritis were associated with focal segmental sclerosis and 10 with focal segmental and focal global sclerosis. In addition, 7 of the 15 cases showed capsular crescents. The clinicopathological correlations indicated that the prognosis in this group is unfavourable when focal global sclerosis and capsular crescents are present, particularly when both occur in the same biopsy specimen.     

Subcellular localisation,protein interactions,and RNA binding of Potato mop-top virus triple gene block proteins

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.     

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.     

Complex formation between hepatitis C virus NS2 and NS3 proteins

Hepatitis C virus (HCV) NS2 and NS3 proteins as well as the NS3 protease cofactor NS4A are essential for the replication of the virus. The presence of in vivo heterodimeric complex between HCV NS2 and NS3 has been suggested by biochemical studies. Detailed characterization of the interactions between these viral proteins is of great importance for better understanding their role in viral replication cycle and represents attractive target for antiviral agents. In this study, we demonstrated in vivo interactions between HCV NS2 and NS3 proteins using an epitope tagging technique. For this purpose NS2, NS3 and NS4A were expressed in fusion with two different tags in Cos7 cells. Immunofluorescence analysis and co-immunoprecipitation with tag-specific antibodies revealed the existence of biologically important NS3/NS4A and NS3/NS2 complexes. Similar complexes were detected also in Huh7 cells infected with Semliki Forest virus vectors expressing NS2 and NS3 or NS23 precursor polyprotein. The formation of complex between NS2 and NS3 was found not to depend on whether the proteins were expressed individually or in form of common precursor. This observation suggests the existence of direct interaction between these two proteins that may have importance for the formation of the whole HCV replication complex.     

Differences in membrane association and sub-cellular distribution between NS2-3 and NS3 of bovine viral diarrhoea virus

The sub-cellular location and mechanism of membrane association of NS3 and NS2-3 polypeptides of bovine viral diarrhoea virus (BVDV) have been examined. Both NS3 and NS2-3 proteins were detected in post-nuclear membrane fractions but not in cytosolic fractions of BVDV infected cells; a proportion of NS3, but not NS2-3, could be dissociated from the membranes with 800 mM KCl or at pH 11. Following extraction with 1% Triton X-114, NS3 was predominantly present in the aqueous phase, but NS2-3 was only recovered in the detergent phase. Confocal microscopy showed that in BVDV infected cells, NS3 and/or NS2-3 co-localise with the endoplasmic reticulum (ER) protein, ERP60, but not Golgi or lysosomal proteins. Sub-cellular fractionation analysis demonstrated that NS2-3 was almost exclusively associated with the rough ER membrane but a significant proportion of NS3 was present in the smooth ER membrane fractions in addition to the rough ER membrane. These differences in the distribution of NS2-3 and NS3 on ER membranes in cells infected with cytopathogenic (CP) strains of BVDV were also observed using confocal microscopy and antibodies that are specific to either NS2 or NS3. This distinct distribution of NS3 and NS2-3 on the ER membrane has revealed a further difference between CP and non-cytopathogenic (NCP) strains of BVDV.     

Neuronal localisation of benzodiazepine receptors in cerebellum.

The cellular localisation of benzodiazepine receptors was studied. Kainic acid induced neuronal lesions (2 x 0.25-2 x 2 micrograms; 2-26 days in rat cerebellum decreased specific binding of [3H]flunitrazepam down to 35% of controls. Specific binding of [3H]flunitrazepam was also decreased (to 80% of controls) in the cerebllum of mutant nervous mouse (nr/nr) where Purkinje cells are degenerated but in the mutant weaver mouse where granule cells are degenerated. These results show that benzodiazepine receptors are located mainly on neurons; both on Purkinje cells and other neurons, but not to a great extent on granule cells.     

Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy

The high genetic variation of hepatitis C virus (HCV) results in rapid selection of drug resistance mutations (DRMs) during monotherapy with direct-acting antivirals (DAAs). It has been proposed that each possible single mutant preexists in infected individuals; however, the levels of preexisting DRMs are too low to be directly quantified in most patients using current techniques. In this study, we evaluated the presence of DRMs in HCV-infected patients treated with the HCV protease inhibitors GS-9256 or GS-9451 as monotherapy using deep sequencing in 137 longitudinal samples from 45 patients. Software was developed to analyze deep-sequencing results with an assay cutoff of 0.25%. No NS3 DRMs that confer resistance to GS-9256 and GS-9451 (R155K, A156T, and D168V/E) were observed in 33 baseline samples at >0.25%. In contrast, these and other substitutions at NS3 positions 155, 156, and 168 were detected in 19/27 patients at day 2 (24 h) and 21/21 at day 4 (84 h) of monotherapy but not in placebo-treated patients. Based on the DRM growth kinetics during drug treatment, pretreated NS3 mutations at amino acids 155, 156, and 168 were estimated on average at 0.025% and 0.015% per genotype 1a and 1b HCV-infected patients, respectively. Relative fitness of the DRM viruses was shown to be significantly lower than the wild type. Deep-sequencing analyses of NS3 protease inhibitor-treated HCV-infected patients suggest a limit of HCV viral load suppression of 3.6 to 3.8 log(10) with NS3 protease inhibitor monotherapy that does not suppress the identified preexisting NS3 DRMs and thus a need for a combination therapy.     

Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells

Although reasons for hepatitis C virus (HCV) persistence are still unknown, specific cellular immune responses appear to influence the pathogenesis and outcome of the infection. Apoptosis of cells infected by viruses may appear suicidal to the viruses that induce programmed cell death of its host. However, apoptosis has been suggested to be a response to virus infection as a mean of facilitating virus dissemination. Annexin V-propidium iodide staining and DNA fragmentation, were used to show that expression of the core, NS3, NS5A, or NS5B protein induces apoptosis in mature dendritic cells. In addition, immunoblotting was used to demonstrate that expression level of p21waf1/cip1 protein decreased in cells expressing one of these HCV proteins. No expression of p53 could be detected and expression of Akt was independent of HCV proteins expression. These results suggest that the effect of these HCV proteins on HCV associated pathogenesis may be linked (at least partially) to its ability to modulate apoptosis pathways in mature dendritic cells.     

Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B-NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser(46) to Ile(60) were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.     

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.     

Variation in the NS3/NS3A gene of bluetongue viruses contained in Culicoides sonorensis collected from a single site in southern California

To determine the variability of the NS3/NS3A gene of field strains of BTV contained in Culicoides sonorensis collected from a single site in California (CA), the NS3/NS3A gene was directly amplified and sequenced from 22 pools of C. sonorensis and compared with those of previously characterized field isolates from CA, as well as to viruses that caused recent outbreaks of bluetongue disease in ruminants in CA. Phylogenetic analysis established that the NS3/NS3A gene of strains of BTV contained in C. sonorensis collected from the site exists as a heterogeneous population. The two most divergent nucleotide sequences of the NS3/NS3A genes of these viruses differed by 2.5% (18 nucleotides). Comparison with the NS3/NS3A gene sequences from viruses that caused recent instances of bluetongue disease in ruminants in CA indicated that BTV strains from different geographic regions can exhibit a higher degree of genetic heterogeneity (up to 6.6%; 0-48 nucleotide differences) than those contained in C. sonorensis collected from a single site.