Publications by year
2018
Pries V, Nöcker C, Khan D, Johnen P, Hong Z, Tripathi A, Keller AL, Fitz M, Perruccio F, Filipuzzi I, et al (2018). Target Identification and Mechanism of Action of Picolinamide and Benzamide Chemotypes with Antifungal Properties.
Cell Chemical Biology,
25(3), 279-290.e7.
Abstract:
Target Identification and Mechanism of Action of Picolinamide and Benzamide Chemotypes with Antifungal Properties
© 2017 Elsevier Ltd Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the identification of chemically versatile benzamide and picolinamide scaffolds with antifungal properties. Chemogenomic profiling and biochemical assays with purified protein identified Sec14p, the major phosphatidylinositol/phosphatidylcholine transfer protein in Saccharomyces cerevisiae, as the sole essential target for these compounds. A functional variomics screen identified resistance-conferring residues that localized to the lipid-binding pocket of Sec14p. Determination of the X-ray co-crystal structure of a Sec14p-compound complex confirmed binding in this cavity and rationalized both the resistance-conferring residues and the observed structure-activity relationships. Taken together, these findings open new avenues for rational compound optimization and development of novel antifungal agents. Rising mortality rates and a limited armamentarium ask for novel, effective antifungal agents. Pries et al. have identified benzamide- and picolinamide-derived compounds that target the fungal lipid-transfer protein Sec14. Intersecting structure-activity relationship data, high-resolution genetics data and the first Sec14p crystal structure in complex with an inhibitor, pave the way for rational evaluation of this compound/target pair for its antifungal potential.
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Lazzaretti D, Bandholz-Cajamarca L, Emmerich C, Schaaf K, Basquin C, Irion U, Bono F (2018). The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity.
Life Science Alliance,
1(5).
Abstract:
The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity
© 2018 Crown copyright. During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar–phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.
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2017
Schellhaus AK, Moreno-Andrés D, Chugh M, Yokoyama H, Moschopoulou A, De S, Bono F, Hipp K, Schäffer E, Antonin W, et al (2017). Developmentally Regulated GTP binding protein 1 (DRG1) controls microtubule dynamics.
Scientific Reports,
7(1).
Abstract:
Developmentally Regulated GTP binding protein 1 (DRG1) controls microtubule dynamics
© 2017 the Author(s). The mitotic spindle, essential for segregating the sister chromatids into the two evolving daughter cells, is composed of highly dynamic cytoskeletal filaments, the microtubules. The dynamics of microtubules are regulated by numerous microtubule associated proteins. We identify here Developmentally regulated GTP binding protein 1 (DRG1) as a microtubule binding protein with diverse microtubule-associated functions. In vitro, DRG1 can diffuse on microtubules, promote their polymerization, drive microtubule formation into bundles, and stabilize microtubules. HeLa cells with reduced DRG1 levels show delayed progression from prophase to anaphase because spindle formation is slowed down. To perform its microtubule-associated functions, DRG1, although being a GTPase, does not require GTP hydrolysis. However, all domains are required as truncated versions show none of the mentioned activities besides microtubule binding.
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Lazzaretti D, Bono F (2017). mRNA localization in metazoans: a structural perspective.
RNA Biology,
14(11), 1473-1484.
Abstract:
mRNA localization in metazoans: a structural perspective
© 2017 the Author(s). Published with license by Taylor. &. Francis Group, LLC © 2017, © Daniela Lazzaretti and Fulvia Bono. Asymmetric localization of mRNAs is a widespread gene regulatory mechanism that is crucial for many cellular processes. The localization of a transcript involves multiple steps and requires several protein factors to mediate transport, anchoring and translational repression of the mRNA. Specific recognition of the localizing transcript is a key step that depends on linear or structured localization signals, which are bound by RNA-binding proteins. Genetic studies have identified many components involved in mRNA localization. However, mechanistic aspects of the pathway are still poorly understood. Here we provide an overview of structural studies that contributed to our understanding of the mechanisms underlying mRNA localization, highlighting open questions and future challenges.
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2016
Lazzaretti D, Veith K, Kramer K, Basquin C, Urlaub H, Irion U, Bono F (2016). The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease.
Nature Structural and Molecular Biology,
23(8), 705-713.
Abstract:
The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease
© 2016 Nature America, Inc. All rights reserved. Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3′-5′ EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3′ untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.
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2014
Bono F (2014). Juggling key players in NMD initiation.
Structure,
22(8), 1074-1075.
Abstract:
Juggling key players in NMD initiation
In this issue of Structure, Melero and colleagues use electron microscopy combined with biochemistry to provide structural insight into the complex between SMG1, SMG8, SMG9, UPF1, and UPF2, elucidating how key players in nonsense-mediated mRNA decay assemble at the initial steps of the process. © 2014 Elsevier Ltd.
Abstract.
2013
Holder T, Basquin C, Ebert J, Randel N, Jollivet D, Conti E, Jékely G, Bono F (2013). Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability.
Biol Direct,
8Abstract:
Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability.
BACKGROUND: Alvinella pompejana is an annelid worm that inhabits deep-sea hydrothermal vent sites in the Pacific Ocean. Living at a depth of approximately 2500 meters, these worms experience extreme environmental conditions, including high temperature and pressure as well as high levels of sulfide and heavy metals. A. pompejana is one of the most thermotolerant metazoans, making this animal a subject of great interest for studies of eukaryotic thermoadaptation. RESULTS: in order to complement existing EST resources we performed deep sequencing of the A. pompejana transcriptome. We identified several thousand novel protein-coding transcripts, nearly doubling the sequence data for this annelid. We then performed an extensive survey of previously established prokaryotic thermoadaptation measures to search for global signals of thermoadaptation in A. pompejana in comparison with mesophilic eukaryotes. In an orthologous set of 457 proteins, we found that the best indicator of thermoadaptation was the difference in frequency of charged versus polar residues (CvP-bias), which was highest in A. pompejana. CvP-bias robustly distinguished prokaryotic thermophiles from prokaryotic mesophiles, as well as the thermophilic fungus Chaetomium thermophilum from mesophilic eukaryotes. Experimental values for thermophilic proteins supported higher CvP-bias as a measure of thermal stability when compared to their mesophilic orthologs. Proteome-wide mean CvP-bias also correlated with the body temperatures of homeothermic birds and mammals. CONCLUSIONS: Our work extends the transcriptome resources for A. pompejana and identifies the CvP-bias as a robust and widely applicable measure of eukaryotic thermoadaptation.
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Author URL.
Grünwald M, Lazzaretti D, Bono F (2013). Structural basis for the nuclear export activity of Importin13.
EMBO Journal,
32(6), 899-913.
Abstract:
Structural basis for the nuclear export activity of Importin13
Importin13 (Imp13) is a bidirectional karyopherin that can mediate both import and export of cargoes. Imp13 recognizes several import cargoes, which include the exon junction complex components Mago-Y14 and the E2 SUMO-conjugating enzyme Ubc9, and one known export cargo, the translation initiation factor 1A (eIF1A). To understand how Imp13 can perform double duty, we determined the 3.6-Å crystal structure of Imp13 in complex with RanGTP and with eIF1A. eIF1A binds at the inner surface of the Imp13 C-terminal arch adjacent and concomitantly to RanGTP illustrating how eIF1A can be exported by Imp13. Moreover, the 3.0-Å structure of Imp13 in its unbound state reveals the existence of an open conformation in the cytoplasm that explains export cargo release and completes the export branch of the Imp13 pathway. Finally, we demonstrate that Imp13 is able to bind and export eIF1A in vivo and that its function is essential. © 2013 European Molecular Biology Organization.
Abstract.
2012
Kinkelin K, Veith K, Grünwald M, Bono F (2012). Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression.
RNA,
18(9), 1624-1634.
Abstract:
Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression
Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation. Published by Cold Spring Harbor Laboratory Press. Copyright © 2012 RNA Society.
Abstract.
2011
Bono F, Gehring NH (2011). Assembly, disassembly and recycling the dynamics of exon junction complexes.
RNA Biology,
8(1).
Abstract:
Assembly, disassembly and recycling the dynamics of exon junction complexes
Efficient gene expression requires that, during their lifetime, mRNAs associate with different sets of RNA binding proteins to form messenger ribonucleoprotein particles (mRNPs). The protein components of mRNPs are essential for the correct post-transcriptional function and regulation of mRNAs. mRNPs are constitutively remodeled during the maturation of the mRNA in the nucleus and downstream steps in the cytoplasm, and can also change depending on the cellular environment. Here we review the current understanding of the biochemical and structural aspects of a central mRNP component and regulator, the exon junction complex (EJC). © 2011 Landes Bioscience.
Abstract.
Grünwald M, Bono F (2011). Structure of Importin13-Ubc9 complex: Nuclear import and release of a key regulator of sumoylation.
EMBO Journal,
30(2), 427-438.
Abstract:
Structure of Importin13-Ubc9 complex: Nuclear import and release of a key regulator of sumoylation
Importin13 (Imp13) is an unusual β-karyopherin that is able to both import and export cargoes in and out of the nucleus. In the cytoplasm, Imp13 associates with different cargoes such as Mago-Y14 and Ubc9, and facilitates their import into the nucleus where RanGTP binding promotes the release of the cargo. In this study, we present the 2.8 Å resolution crystal structure of Imp13 in complex with the SUMO E2-conjugating enzyme, Ubc9. The structure shows an uncommon mode of cargo-karyopherin recognition with Ubc9 binding at the N-terminal portion of Imp13, occupying the entire RanGTP-binding site. Comparison of the Imp13-Ubc9 complex with Imp13-Mago-Y14 shows the remarkable plasticity of Imp13, whose conformation changes from a closed ring to an open superhelix when bound to the two different cargoes. The structure also shows that the binding mode is compatible with the sumoylated states of Ubc9. Indeed, we find that Imp13 is able to bind sumoylated Ubc9 in vitro and suppresses autosumoylation activity in the complex. © 2011 European Molecular Biology Organization | all Rights Reserved.
Abstract.
2010
Buchwald G, Ebert J, Basquin C, Sauliere J, Jayachandran U, Bono F, Le Hir H, Conti E (2010). Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex.
Proceedings of the National Academy of Sciences of the United States of America,
107(22), 10050-10055.
Abstract:
Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex
In mammals, Up-frameshift proteins (UPFs) form a surveillance complex that interacts with the exon junction complex (EJC) to elicit nonsense-mediated mRNA decay (NMD). UPF3b is the component of the surveillance complex that bridges the interaction with the EJC. Here, we report the 3.4 Å resolution crystal structure of a minimal UPF3b-EJC assembly, consisting of the interacting domains of five proteins (UPF3b, MAGO, Y14, eIF4AIII, and Barentsz) together with RNA and adenylyl-imidodiphosphate. Human UPF3b binds with the C-terminal domain stretched over a composite surface formed by eIF4AIII, MAGO, and Y14. Residues that affect NMD when mutated are found at the core interacting surfaces, whereas differences between UPF3b and UPF3a map at peripheral interacting residues. Comparison with the binding mode of the protein PYM underscores how a common molecular surface of MAGO and Y14 recognizes different proteins acting at different times in the same pathway. The binding mode to eIF4AIII identifies a surface hot spot that is used by different DEAD-box proteins to recruit their regulators.
Abstract.
Bono F, Cook AG, Grünwald M, Ebert J, Conti E (2010). Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13.
Molecular Cell,
37(2), 211-222.
Abstract:
Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13
Mago and Y14 are core components of the exon junction complex (EJC), an assembly central to nonsense-mediated mRNA decay in humans and mRNA localization in flies. The Mago-Y14 heterodimer shuttles between the nucleus, where it is loaded onto specific mRNAs, and the cytoplasm, where it functions in translational regulation. The heterodimer is imported back into the nucleus by Importin 13 (Imp13), a member of the karyopherin-β family of transport factors. We have elucidated the structural basis of the Mago-Y14 nuclear import cycle. The 3.35 Å structure of the Drosophila Imp13-Mago-Y14 complex shows that Imp13 forms a ring-like molecule, reminiscent of Crm1, and encircles the Mago-Y14 cargo with a conserved interaction surface. The 2.8 Å structure of human Imp13 bound to RanGTP reveals how Mago-Y14 is released in the nucleus by a steric hindrance mechanism. Comparison of the two structures suggests how this unusual karyopherin might function in bidirectional nucleocytoplasmic transport. © 2010 Elsevier Inc. All rights reserved.
Abstract.
2007
Cook A, Bono F, Jinek M, Conti E (2007).
Structural biology of nucleocytoplasmic transport.Abstract:
Structural biology of nucleocytoplasmic transport
Abstract.
2006
Bono F, Ebert J, Lorentzen E, Conti E (2006). The Crystal Structure of the Exon Junction Complex Reveals How it Maintains a Stable Grip on mRNA.
Cell,
126(4), 713-725.
Abstract:
The Crystal Structure of the Exon Junction Complex Reveals How it Maintains a Stable Grip on mRNA
The exon junction complex (EJC) plays a major role in posttranscriptional regulation of mRNA in metazoa. The EJC is deposited onto mRNA during splicing and is transported to the cytoplasm where it influences translation, surveillance, and localization of the spliced mRNA. The complex is formed by the association of four proteins (eIF4AIII, Barentsz [Btz], Mago, and Y14), mRNA, and ATP. The 2.2 Å resolution structure of the EJC reveals how it stably locks onto mRNA. The DEAD-box protein eIF4AIII encloses an ATP molecule and provides the binding sites for six ribonucleotides. Btz wraps around eIF4AIII and stacks against the 5′ nucleotide. An intertwined network of interactions anchors Mago-Y14 and Btz at the interface between the two domains of eIF4AIII, effectively stabilizing the ATP bound state. Comparison with the structure of the eIF4AIII-Btz subcomplex that we have also determined reveals that large conformational changes are required upon EJC assembly and disassembly. © 2006 Elsevier Inc. All rights reserved.
Abstract.
2004
Bono F, Ebert J, Unterholzner L, Güttler T, Izaurralde E, Conti E (2004). Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex.
EMBO Reports,
5(3), 304-310.
Abstract:
Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex
The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago-Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago-Y14. Here we show that PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago-Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD. © 2004 European Molecular Biology Organization.
Abstract.
1999
Sabile A, Perlemuter G, Bono F, Kohara K, Demaugre F, Kohara M, Matsuura Y, Miyamura T, Bréchot C, Barba G, et al (1999). Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates.
Hepatology,
30(4), 1064-1076.
Abstract:
Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates
Several lines of evidence suggest that hepatitis C virus (HCV) core protein may modulate cellular transduction signals and alter lipid metabolism. We have investigated the binding of HCV core protein to cellular proteins by combining 2 yeast hybrid, confocal, and surface plasmon resonance assays. Our results show the direct binding of the viral protein to apolipoprotein AII (apoAII) and map the interaction domain to the C-terminal of HCV core protein. To investigate the biological relevance of the interaction between HCV core and lipid metabolism, we took advantage of the well-established increase in apoAII expression caused by fibrates in HepG2 cells. After fenofibric acid treatment, we show a parallel increase in apoAII and core protein secretion, this effect being abolished by brefeldin A. Our study identifies apoAII as one of the cellular targets for HCV core protein. We also show that the intervention of fenofibric acid in cellular lipid metabolism directly affects the expression pattern of HCV core protein.
Abstract.
1997
Bruno S, Silini E, Crosignani A, Borzio F, Leandro G, Bono F, Asti M, Rossi S, Larghi A, Cerino A, et al (1997). Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
Hepatology,
25(3), 754-758.
Abstract:
Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study
A prospective study was performed to establish whether infection with specific hepatitis C virus (HCV) genotypes was associated with an increased risk of development of hepatocellular carcinoma (HCC) in cirrhosis. A cohort of 163 consecutive hepatitis C virus antibody (anti-HCV)-positive cirrhotic patients was prospectively evaluated for the development of HCC at 6-month intervals by ultrasound (US) scan and α-fetoprotein (AFP) concentration. HCV genotypes were determined according to Okamoto. Risk factors associated with cancer development were analyzed by univariate and multivariate statistics. At enrollment, 101 patients (62%) were infected with type 1b, 48 (29.5%) were infected with type 2a/c, 2 (1.2%) were infected with type 3a, 1 (0.6%) was infected with type 1a, 3 (1.8%) had a mixed-type infection, and, in 8 patients (4.9%), genotype could not he assigned. After a 5- to 7-year follow- up (median, 68 months), HCC developed in 22 of the patients, 19 infected with type 1b and 3 with type 2a/c (P <. 005). Moreover, HCC developed more frequently in males (P <. 01), patients with excessive alcohol intake (P <. 01), those over 60 years of age (P <. 02), and in patients who did not receive interferon treatment (P <. 02). Multivariate analysis showed that type lb was the most important risk factor associated with tumor development (odds ratio 6.14, 1.77-21.37 95% confidence interval). Other independent risk factors were older age and male sex. Cirrhotic patients infected with HCV type 1b carry a significantly higher risk of developing HCC than patients infected by other HCV types. The latter may require a less intensive clinical surveillance for the early detection of neoplasia.
Abstract.
1996
Silini E, Bottelli R, Asti M, Bruno S, Candusso ME, Brambilla S, Bono F, Iamoni G, Tinelli C, Mondelli MU, et al (1996). Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a case-control study.
Gastroenterology,
111(1), 199-205.
Abstract:
Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a case-control study
Background and Aims: Viral genotypes have been associated with different severity and outcome of hepatitis C virus (HCV)-related liver disease. The aim of this study was to determine whether HCV genotypes may influence the cirrhosis-related risk of the development of hepatocellular carcinoma (HCC). Methods: Three groups of patients were studied: 593 patients with chronic hepatitis, 166 patients with HCC and cirrhosis, and 219 patients with cirrhosis but without HCC. A cross-sectional study of frequency distribution and a case-control analysis were performed. HCV genotypes were detected according to Okamoto. Results: HCV type lb infection was more prevalent among patients with HCC compared with patients with cirrhosis but without HCC (P < 0.01) and chronic hepatitis (P < 0.001). Age, male sex, and HCV type 1b significantly influenced the risk of cancer in cirrhosis by univariate analysis. A pairwise comparison performed on 162 patients with HCC and an equal number of patients with cirrhosis matched by age, sex, and Child's class showed that HCV type 1b was independently associated with HCC (odds ratio, 1.7; P = 0.026). Conclusions: HCV type 1b is overrepresented in patients with cirrhosis and HCC and significantly influences the risk of HCC in cirrhosis, independent of sex, age, and Child's class.
Abstract.
1995
Baldanti F, Silini E, Sarasini A, Talarico CL, Stanat SC, Biron KK, Furione M, Bono F, Palu G, Gerna G, et al (1995). A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.
Journal of Virology,
69(2), 796-800.
Abstract:
A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate
Multiple human cytomegalovirus (HCMV) strains frequently coexist in patients with AIDS, and chronic ganciclovir treatment may favor the emergence of ganciclovir-resistant viral mutants. We report the molecular and biochemical characterization of a HCMV ganciclovir-resistant strain (VR3480) previously recovered from a patient with AIDS who was undergoing multiple courses of ganciclovir treatment (G. Gerna, F. Baldanti, M. Zavattoni, A. Sarasini, E. Percivalle, and M. G. Revello, Antiviral Res. 19:333-345, 1992). Ganciclovir resistance of strain VR3480 was related to impaired ability to monophosphorylate the drug, as indicated by the finding that ganciclovir phosphorylation values for VR3480 were 30% of those shown by the HCMV reference strain AD169 in an in vitro activity assay. Sequencing of the UL97 gene of VR3480, which encodes the viral kinase responsible for ganciclovir phosphorylation, showed an in-frame deletion of three nucleotides resulting in the loss of a leucine at position 595 in the polypeptide. Mutant VR3480 UL97 DNA was able to transfer resistance to the AD169 strain in marker rescue experiments. Analysis of virus isolates and blood polymorphonuclear leukocyte samples spanning the 2-year follow-up period of the patient showed that ganciclovir-resistant strain VR3480 arose ex novo during prolonged antiviral treatment and accounted for the majority of virus load circulating in blood during the period of clinical resistance to ganciclovir treatment.
Abstract.
Silini E, Bono F, Cividini A, Cerino A, Bruno S, Rossi S, Belloni G, Brugnetti B, Civardi E, Salvaneschi L, et al (1995). Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities.
Hepatology,
21(2), 285-290.
Abstract:
Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities
Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription-polymerase chain reaction (RTPCR) in 341 consecutive anti-HCV-positive patients. of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations ≤5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P
Abstract.
Silini E, Bono F, Cividini A, Cerino A, Maccabruni A, Tinelli C, Bruno S, Bellobuono A, Mondelli MU (1995). Molecular epidemiology of hepatitis C virus infection among intravenous drug users.
Journal of Hepatology,
22(6), 691-695.
Abstract:
Molecular epidemiology of hepatitis C virus infection among intravenous drug users
Background/Aims: the clinico-pathologic features of hepatitis C virus infection intravenous drug users are different from those found in other hepatitis C virus-infected patients. Our airm was to test whether specific viral variants circulate within this particular patient population. Methods: We studied the distribution of hepatitis C virus genotypes in 90 drug addicts and 484 controls, according to the method described by Okamoto. Results: Hepatitis C virus type 1a and 3a infections were more frequent among intravenous drug users than in 125 age-matched controls (48.8% and 21.1% vs 17.6% and 11.2%), accounting for the majority of infections in intravenous drug users. Analysis of hepatitis C virus genotypes according to age showed that, in the general population, hepatitis C virus types 1a and 3a were more prevalent among patients younger than 40 years of age than in older individual (17.6% and 11.2% vs. 1.4% and 0.6%). Conclusions: These findings suggest that hepatitis C virus types 1a and 3a were recently introduced in Italy, presumably via needle-sharing among intravenous drug users, and from this reservoir they are extending to the general population, particularly among younger subjects. © 1995.
Abstract.
1994
Mondelli MU, Cerino A, Bono F, Cividini A, Maccabruni A, Arico M, Malfitano A, Barbarini G, Piazza V, Minoli L, et al (1994). Hepatitis C virus (HCV) core serotypes in chronic HCV infection.
Journal of Clinical Microbiology,
32(10), 2523-2527.
Abstract:
Hepatitis C virus (HCV) core serotypes in chronic HCV infection
Recently, two distinct hepatitis C virus (HCV) serologic types have been identified on the basis of amino acid variations in the core region. The two serologic types can readily discriminate between genotypes I-II-V (serotype 1) and III-IV (serotype 2), according to the Okamoto classification. We compared HCV core serotyping with genotyping with sera from 363 anti-HCV- positive patients (309 HCV RNA positive by PCR) using a synthetic core peptide-based enzyme immunoassay and PCR amplification of core region sequences with type-specific primers, respectively. Serologic responses to HCV serotypes were successfully identified in 164 (45%) patients, of whom 153 were viremic. Eighty-nine patients had evidence of exposure to serotype 1: 8 of these were infected with genotype I, 50 were infected with genotype II, 2 were infected with genotype III, 7 were infected with genotype V, 13 had infections with mixed genotypes, 3 were infected with an indeterminate genotype, and 6 were nonviremic. Seventy-four patients had been exposed to serotype 2: 64 were infected with genotype III, 3 were infected with mixed genotypes, 2 were infected with an indeterminate genotype, and 5 were nonviremic. The serum of one patient, infected with genotype III, showed reactivity to both serotypes. Comparative evaluation of HCV core region serotyping and genotyping with sera from 294 viremic patients infected with a known HCV genotype showed a remarkable concordance between HCV core region genotyping and serotyping, with only 2 apparently discordant serum samples (both from patients with genotype III infection) of 148 (1.4%) successfully serotyped samples. Serotype 1 infection was more frequently observed in patients with overt chronic liver disease and accounted for all successfully serotyped samples from intravenous drug abusers. In contrast, serotype 2 was more prevalent in subjects with biochemically silent HCV infection (alanine aminotransferase,
Abstract.
Aricò M, Maggiore G, Silini E, Bono F, Viganò C, Cerino A, Mondelli MU (1994). Hepatitis C virus infection in children treated for acute lymphoblastic leukemia.
Blood,
84(9), 2919-2922.
Abstract:
Hepatitis C virus infection in children treated for acute lymphoblastic leukemia
We studied 102 consecutive subjects after their completion of acute lymphoblastic leukemia (ALL)-directed chemotherapy, for evidence of hepatitis C virus (HCV) infection by enzyme immunoassay 2 and 3, second generation recombinant immunoblot assay and reverse transcription-polymerase chain reaction (PCR) for detection of circulating HCV-RNA. Forty-four patients (43%) had evidence of exposure to HCV; 30 of these were anti-HCV+. of the 23 patients who were positive for both anti-HCV and HCV-RNA, 16 (69%) had a moderate increase in serum alanine aminotransferase (ALT) activity without clinical signs of liver disease. Fourteen patients were seronegative despite the presence of HCV-RNA in the serum. The proportion of different HCV genotypes was not significantly different from other anti-HCV+ patient groups. Although half of the patients with genotype III had normal ALT value, patients with normal ALT levels were represented in all genotype groups. Our study documents the prevalence of HCV infection in childhood ALL survivors, which is responsible for the majority of cases of non-B chronic liver disease in these patients. Whereas serologic screening identifies over 70% of patients with ongoing HCV infection, real HCV infection may be present even in the absence of a detectable humoral immune response to the virus. Based on this observation, determination of HCV-RNA by PCR should be recommended in patients in prolonged remission even if they test negative on serological assay. Normal ALT levels do not exclude the presence of HCV infection because the values were repeatedly normal in over half of our viremic patients.
Abstract.
1993
Magrassi L, Butti G, Silini E, Bono F, Paoletti P, Milanesi G (1993). The expression of genes of the steroid-thyroid hormone receptor superfamily in central nervous system tumors.
Anticancer Research,
13(4), 859-866.
Abstract:
The expression of genes of the steroid-thyroid hormone receptor superfamily in central nervous system tumors
We studied the expression of ten genes (encoding the receptors for glucocorticoid, mineralocorticoid, progesterone, androgen, estrogen, thyroid, retinoid acid, vitamin D) belonging to the steroid-thyroid hormone receptor superfamily (STRS) in 12 neuroepithelial tumors, 12 meningiomas and 2 human glioblastoma cell lines. Our method, based on the polymerase chain reaction, allowed the simultaneous amplification of cDNAs of the STRS genes. On average, 7 STRS genes were simultaneously expressed in each sample. Our study indicates that many STRS genes are commonly coexpressed in human CNS tumors. The importance of our results for the ongoing and proposed hormonal treatment trials is discussed.
Abstract.
Silini E, Bono F, Cerino A, Piazza V, Solcia E, Mondelli MU (1993). Virological features of hepatitis C virus infection in hemodialysis patients.
Journal of Clinical Microbiology,
31(11), 2913-2917.
Abstract:
Virological features of hepatitis C virus infection in hemodialysis patients
The clinical and epidemiological relevance of circulating antibodies to hepatitis C virus (HCV) in hemodialysis patients is uncertain, since clinical signs of infection are often mild or absent, with alanine aminotransferase (ALT) values that are virtually always normal, and liver biopsies are only rarely performed. Determination of HCV RNA in serum is therefore critical for distinguishing chronic HCV infection from previous exposure to the virus. We studied HCV viremia by reverse transcription polymerase chain reaction (RT- PCR) in the 5'-noncoding region of the viral genome in 77 dialysis patients who were screened for anti-HCV by a second-generation enzyme-linked immunosorbent assay (the enzyme immunoassay II; Ortho HCV, 2nd generation, Ortho Diagnostic Systems Raritan, N.J.) and a second-generation recombinant immunoblot assay (Chiron Corporation and Ortho Diagnostic Systems) and prospectively evaluated for ALT elevations over a period of 5 years. of 77 patients tested, 29 (38%) had active infection as shown by a positive PCR assay result, and of these, 26 were anti-HCV positive. Although a good correlation was found between circulating anti-HCV and HCV RNA in serum, 10 (28%) of 36 anti-HCV-positive patients were HCV RNA negative by PCR, suggesting either low levels of viremia or past exposure to HCV and subsequent recovery. On the other hand, 3 (7.3%) of 41 anti-HCV-negative patients had HCV RNA in their sera, indicating seronegative HCV infection. The ALT level had no predictive value for HCV infection, because it was repeatedly normal in 18 (62%) of 29 viremic patients. HCV genotyping was also performed and indicated that all four known genotypes of HCV were present in our group. In conclusion, serological assays are reliable for detecting exposure to HCV in hemodialysis patients; however, direct identification of the viral genome is required to document current infection.
Abstract.
1992
Magrassi L, Bono F, Milanesi G, Butti G (1992). Vitamin D receptor expression in human brain tumors. Journal of Neurosurgical Sciences, 36(1), 27-30.
