For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.
The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.Despite the introduction of blood-screening tests ϳ10 years ago, hepatitis C virus (HCV) is still the major cause of bloodborne chronic hepatitis, with nearly 200 million infected people worldwide. HCV infection often evolves into a chronic disease, which can lead to liver dysfunction and hepatocellular carcinoma. Current therapeutic regimens based on alpha interferon (IFN-␣) and the nucleoside analog ribavirin are only partially effective and are limited by the adverse effects of both agents (50). Given the high prevalence of this disease, developing new treatments is a major public health objective. Similarly to human immunodeficiency virus (HIV) research, most efforts to develop antiviral agents for HCV have focused on the inhibition of key viral enzymes, serine protease, helicase, and polymerase (2).The most extensively studied HCV target has been the NS3-4A serine protease, a heterodimeric enzyme comprising the N-terminal domain of the NS3 protein (amino acids 1 to 180) and the small hydrophobic NS4A protein (3). This protease cleaves the viral polyprotein at four junctions (NS3/ NS4A, NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A), and its activity is necessary for viral replication (24). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/ NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (4, 14, 28, 47). X-ray crystallography (20, 35, 51) and nuclear magnetic resonance (NMR) spectroscopy (1, 36) have shown that the NS3-4A structure is similar to that of other chymotrypsin-like serine proteases, with two domains, both composed of a -barrel and two short ␣-helices. The catalytic triad comprises histidine 57, aspartate 81, and serine 139 and is located between the two domains. The central region of NS4A is an integral part of t...
Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low-to intermediate-risk HPV.Human papillomaviruses (HPVs) are the most studied members of the family designated Papillomaviridae, DNA viruses sharing an unenveloped icosahedral structure as well as the basic organization and replication strategy of the circular, double-stranded, 8-kb DNA genome. To date more than 80 genotypes have been completely characterized, and a number of partially characterized isolates, probably representing novel genotypes, are presently under investigation (43). HPVs can be divided into 3 of the 7 supergroups described for all papillomaviruses (6); supergroup A is associated with genital disease, and a subset of its members is found in the vast majority of genital cancers (1, 43).HPV infection is extremely frequent throughout the world. Most of these infections are transient (15), although the virus can persist in a very limited number of infected cells, and do not reach clinical observation. In a small proportion of nonimmune-suppressed subjects and in the majority of immunesuppressed individuals HPV infection can persist for years in clinically evident lesions. During the last few years, different molecular strategies have been employed for detecting and typing novel HPVs, mostly by using PCR amplification and general primers for the conserved L1 or E1 viral regions. The widespread use of these methods (14,28,32,40) is leading to the discovery of a rising number of HPV genotypes (4,7,11,12,18,(20)(21)(22) whose roles as human pathogens remain to be established.Recently, we were interested in identifying unknown HPV genotypes infecting patients with gynecologic or dermatologic lesions and adopted a molecular method that allowed the identification of potentially n...
Purpose Recently, there was an increasing interest on the use of ancient grains because of their better health-related composition. The aim of this study was to evaluate in healthy human subjects the antioxidative and diabetes-preventive properties of ancient KAMUT ® khorasan wheat compared to modern wheat. Methods The study was a randomized, non-blind, parallel arm study where the biochemical parameters of volunteers with a diet based on organic whole grain KAMUT ® khorasan products, as the only source of cereal products were compared to a similar replacement diet based on organic whole grain modern durum wheat products. A total of 30 healthy volunteers were recruited and the intervention period lasted 16 weeks. Blood analyses were performed before and after the diet intervention. The effect of KAMUT ® khorasan products on biochemical parameters was analyzed by multiple quantile regression adjusted for age, sex, physical activity and BMI compared to data at baseline. Results Subjects receiving KAMUT ® khorasan products showed a significantly greater decrease of fat mass (b = 3.7%; CI 1.6-5.5; p = 0.042), insulin (b = 2.4 µU/ml; CI 0.2-4.2; p = 0.036) and a significant increase of DHA (b = − 0.52%; CI − 1.1 to − 0.12; p = 0.021). Conclusions Our study provides evidence that a substitution diet with KAMUT ® khorasan wheat products can reduce some markers associated to the development of type-2 diabetes compared to a diet of modern wheat.
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