Biomedical subjects
Publications and source records attributed to M Melegari.
A series of N-(4-methoxy, 4-fluoro, 4-trifluoromethyl and 4-nitrobenzoyl)-L-amino acids was synthesized and their inhibitory activity towards bovine lens aldose reductase (ALR2) was tested.
Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.
The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.
It is now well recognized that mutations in the hepatitis B virus (HBV) genome occur during the natural course of chronic viral infection. Regions of the viral genome that are frequently affected by such mutations, rearrangements, and/or deletions generally involve the precore promoter, precore, and core as well as the preS gene regions. However, little is known regarding the biologic consequences of these mutations on the functional properties of the variant viral strains with respect to effects on viral replication. In this study, we investigated the functional significance of precore promoter and precore gene mutations that reduce or abolish the synthesis of hepatitis B e antigen (HBeAg). We found that precore promoter mutations diminished the expression of HBeAg but did not affect the synthesis of pregenomic RNA. However, these precore mutations were associated with a modest increase in HBV replication. In contrast, a naturally occurring mutant that carries a termination codon in position 28 of the precore open reading frame demonstrated increased encapsidation of pregenomic mRNA into nucleocapsid particles. Consequently, this variant viral strain demonstrated a substantial increase in the level of viral replication compared to "wild-type" HBV and other precore promoter mutant viral strains. These studies suggest that substitutions in the precore promoter and precore gene not only alter the synthesis of HBeAg but also affect the level of viral replication.
Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid-based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA-based immunization techniques to stimulate broad-based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the host's cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.
Hepadnaviruses encode two core-related open reading frames. One directs the synthesis of the p21 core protein, which subsequently becomes a structural component of the viral nucleocapsid. The other produces a p25 precore protein that is targeted by a signal peptide to a cell secretory pathway where N-terminal processing will create a p22 species. This molecule will be further modified at the C-terminal region to generate p17, and the truncated protein is secreted from the cell as hepatitis B e antigen (HBeAg). The function of the precore gene in the biology of hepadnaviruses is unknown. We found that ablation of the precore gene resulted in the generation of a hepatitis B virus (HBV) species with a high-replication-level phenotype. More important, expression in trans of physiologic levels of p25 restored viral replication to wild-type levels. Moreover, transient or stable overexpression of the precore gene resulted in striking inhibition of HBV replication. The molecular species responsible for this viral inhibitory effect was identified as the p22 nonsecreted HBeAg precursor protein. By sucrose gradient sedimentation analysis, we determined that expression of p22 leads to the formation of nucleocapsids similar to those made with wild-type p21 core protein. Immunoprecipitation experiments revealed that the p21 and p22 physically interact and form hybrid nucleocapsid structures devoid of pregenomic viral RNA. These experiments suggest that expression of the precore gene may be important in the regulation of HBV replication and describe a possible molecular mechanism(s) for this effect.
Naturally occurring deletions in the hepatitis B virus pre-S1 domain have been frequently found during persistent viral infection. In this study we have investigated the functional properties of a mutant viral genome that carries an in-frame deletion of 183 nucleotides in the pre-S1 region. This deletion removes the promoter of the small envelope gene. Transfection into human hepatocellular carcinoma cells of a replication-competent construct containing this deletion resulted in an increase of intermediate DNA replicative forms compared to those produced by wild-type hepatitis B virus. Northern blot analysis revealed that such cells lack the 2.1-kb transcripts encoding the small envelope protein and that hepatitis B surface antigen was absent as well. Furthermore, nucleocapsids containing the genome with pre-S1 deleted were not secreted, and the deleted large envelope protein was retained with the cytoplasm and exhibited a perinuclear pattern of distribution. However, coexpression with the small envelope protein was sufficient to restore virion secretion and to change the cellular distribution of the deleted large envelope protein. In addition, the creation of point mutations that prevent the synthesis of large or small envelope proteins also inhibited viral secretion and led to increased levels of hepatitis B virus intermediate replicative forms within the cell. These studies suggest that naturally occurring viral mutants with pre-S1 deletions involving the promoter region of the small envelope gene will generate a deleted large envelope protein that is retained in the endoplasmic reticulum, resulting in the accumulation of nucleocapsids containing viral DNA; transcomplementation with the wild-type small envelope protein will allow mutant virion secretion to occur.
A number of 1H-benzoimidazol-2-ylamine and of 1-methyl-1H-benzoimidazol-2-ylamine derivatives were synthesized and the crystal and molecular structure of N-[4-(2-amino-benzoimidazole-1-sulfonyl)-phenyl] acetamide was determined by X-ray diffraction analysis. The compounds obtained were investigated for antimicrobial and genotoxic activities.
Chronic hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. We have recently described HBV and woodchuck hepatitis virus (WHV) dominant negative (DN) core mutants that were capable of inhibiting wild-type viral replication by 95%. These mutants may represent a potent class of antiviral agents that act as "intracellular immunogens." To facilitate their potential use in animal model systems, we now have studied the duck HBV (DHBV) and placed the DN mutant constructs in recombinant retroviral and adenoviral expression vectors. Transient expression of the DHBV molecular equivalent of the WHV and HBV DN constructs inhibited wild-type DHBV replication by 98%. Recombinant retroviral and adenoviral vectors containing the HBV and DHBV DN complementary DNAs (cDNAs) were used to transiently and stably transduce hepatoma-derived cell lines constitutively expressing replicating wild-type virus. These investigations show that the DN core mutants were powerful inhibitors of HBV and DHBV replication when delivered intracellularly and appear as promising antiviral agents for gene therapy of persistent viral infection of the liver.
Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus. Acute and chronic HBV infection causes significant liver diseases such as acute hepatis, fulminant hepatitis and chronic active hepatitis that may lead to liver cirrhosis and the development of hepatocellular carcinoma. The use of molecular biological techniques has substantially improved our understanding of the HBV life cycle. In this review, we discuss recent advances that have contributed to a better understanding of HBV biology. Recent studies in the understanding of the life cycle of HBV such as viral entry, replication, transcriptional regulation, viral regulatory proteins, viral assembly and secretion, and nucleic acid based approaches to antiviral therapy will be emphasized. These advances in molecular biology and relationship to clinical disease will be instrumental in developing effective therapeutic approaches for the estimated 300 million individuals worldwide chronically infected with HBV.
We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.
Deletion mutants of hepatitis B virus (HBV) pre-S proteins were detected in serum in 2 of 10 (20%) individuals with chronic hepatitis B infection following the initiation of interferon treatment. The size of these deletions was up to one-half of the entire pre-S1 region. In vivo, all HBV deletion mutants were found to coexist with a full-length "wild-type" viral genome. The functional properties of a HBV-deleted mutant were studied in detail and revealed a stop codon in the pre-S2 open reading frame in all 20 of the clones sequenced. Several of the deleted mutants produced low-level HBsAg in culture supernatants compared to wild-type virus due to a putative loss of transcription factor binding sites. Transfection experiments in human hepatoma cells (HuH-7) demonstrated that the polymerase gene function was not affected by the large pre-S1 deletions and mutant viral genomes were capable of replication. However, secretion of incapsidated mutant viral genomes was blocked in HuH-7 cells. Cotransfection studies with a plasmid expressing only the HBV pre-S1, pre-S2, and S proteins resulted in complete restoration of viral particle secretion. Our findings suggest that an in vivo trans-complementation phenomenon would have had to occur to permit secretion from the liver into serum of the nucleocapsids containing these deleted viral genomes.
We report on the analysis of HBV transcription in peripheral blood mononuclear cells of chronically infected patients by polymerase chain reaction amplification. Our results suggest that in these cells gene expression occurs either as pregenomic or subgenomic transcripts.
To study the causes of residual posttransfusion hepatitis, serum from implicated donors was tested by PCR for the presence of HCV RNA. Of 20 anti HCV negative donors, 4 were HCV RNA positive and thus, infective. The results suggest that higher-level investigations are necessary for prospective donors who present blood enzyme abnormalities or other questionable characteristics.
The absence of detectable anti-HBc antibodies in some hepatitis B virus (HBV) infected patients may be due to altered core-protein (HBc) sequences. To investigate this possibility we sequenced the pre-C/C-region of HBV isolated from 12 juvenile cancer patients who incurred a nosocomial infection of HBV during chemotherapy but did not develop anti-HBc antibodies or acute cytolytic episodes. The sequences demonstrated the highest sequence homology to the pre-C/C region of a previously cloned HBV genome (subtype ayw) and no deletions or striking mutations were detected. Up to 7 years after infection almost all the survivors developed low titers of anti-HBc antibodies but no clinical signs of hepatic damage. These results suggest that chemotherapy may induce a tolerance status to HBcAg, the most immunogenic HBV protein.
A group of blood donors involved in post-transfusion hepatitis was investigated for the presence of the anti-HCV antibody and of HCV RNA as a more direct infection marker. RNA was extracted from serum, reverse transcribed and amplified using primers which belonged to the non structural region. The amplified product of the PCR reaction was 582 base pairs. Seven (25.9%) of the 27 blood donors examined were found anti-HCV-positive by ELISA; five (71.4%) of these were HCV RNA positive. Among the 20 anti-HCV-negative blood donors, four (20.0%) were HCV RNA positive. ALT levels were below 45 UI/l in 18 donors, while the other nine had ALTs over the limit accepted for transfusion. The anti-HCV-negative HCV RNA-positive blood donors had normal ALTs. Our study offers a direct explanation for the substantial proportion of residual cases of anti-HCV-positive post-transfusion hepatitis and suggests the necessity of creating a register of blood donors who have at some time presented blood enzyme abnormalities and for whom second level investigations such as HCV RNA should be used.
Genotoxic properties of essential oils from Anthemis nobilis L., Artemisia dracunculus L., Salvia officinalis L., Salvia sclarea L., Satureja hortensis L., Satureja montana L., Thymus capitatus L., Thymus citriodorus Schreb., Thymus vulgaris L., Citrus bergamia Risso, were studied with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay. The essential oil of Artemisia dracunculus L. "Piemontese" turned out to be active in the rec-assay but not in the Salmonella test. DNA-damaging activity was demonstrated to be due to the estragol component of the oil. Advantages of the combined use of these two short-term microbial assays in genotoxic studies are discussed.