The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutatio... more The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.
We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum sa... more We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection.
Nietzsche was the first major philosopher contesting Darwin's principle of «struggle for existenc... more Nietzsche was the first major philosopher contesting Darwin's principle of «struggle for existence» as the motor for evolution. Nietzsche opposed to this principle that of «struggle for power». Nietzsche did not deny natural selection, but thought that selection acted essentially on those transformations produced by the struggle for power. The testable hypothesis proposed by Nietzsche is that the victory in the struggle for power does not belong to the strongest, most fortunately constituted individual but rather to the more abundant, meanest individuals (Table ). However, science has not provided a mechanistic formulation of the Nietzschean concept of power relationships capable of undertaking the challenge of some experimental exploration. In this essay we propose that there has been a theoretical formulation, amenable to experimental analysis, which provides a test of power relationships among components of a population of individuals. We refer to the self-replication model of M. Eigen formulated to describe early replicons at the origin of life, and which has found an application in the understanding of RNA viruses. We describe experimental observations on competition among RNA genomes, and we relate the findings to Nietzschean power relationships.
Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies ... more Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previ...
Since January 2020 Elsevier has created a COVID-19 resource centre with free information in Engli... more Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre -including this research content -immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (H... more The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (HRGP) was determined by in situ hybridization. During normal development of roots and leaves, the expression of the gene was transient and particularly high in regions initiating vascular elements and associated sclerenchyma. Its expression was also associated with the differentiation of vascular elements in a variety of other tissues. The gene encoded an HRGP that had been extracted from the cell walls of maize suspension culture cells and several other embryonic and post-embryonic tissues. The gene was present in one or two copies in different varieties of maize and in the related monocots teosinte and sorghum. A single gene was cloned from maize using a previously characterized HRGP cDNA clone . Plant MOI. Biol. 11, 483-4931. In addition to the coding sequences for the HRGP and an N-terminal signal sequence, the gene contained a single intron in the nontranslated 3' end.
Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the... more Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. Picornavirus IRES are organized in structural domains, in which the terminal stem–loops participate in functional RNA–protein interactions. However, the mechanistic role performed by the central domain during internal initiation has not been elucidated yet. Here we show that the foot-and-mouth-disease virus IRES contains a structural motif that serves in vitro as substrate for the Synechocystis sp. RNase P ribozyme, a structure-dependent endonuclease that participates in tRNA precursor processing. Recognition of the IRES substrate was dose dependent, required high magnesium concentration, and resulted in the formation of cleavage products with 5′ phosphate and 3′ hydroxyl ends. Mapping of the core recognition motif indicated that it overlapped with the apical region of the central domain. Two IRES constructs containing nucleotide substitutions...
Previous studies have identified a set of highly phosphorylated proteins of 23-25 kDa accumulated... more Previous studies have identified a set of highly phosphorylated proteins of 23-25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants. During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23-25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABAdeficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos. These results indicate that the 23-25 kDa protein set is a new member of the recently described class of proteins involved in generalized plant ABA responses. The different pattern of expression for the ABA-regulated 23-25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.
In the course of a study conducted from 1992 through 1994 of the efficacy of screening blood dono... more In the course of a study conducted from 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. Methods. Of 222 of the surgeon's patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. Results. The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusionassociated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P Ͻ 0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of a phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. Conclusions. Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery. (
We have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This g... more We have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction, rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vpl mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vpl contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.
Previously, we described two RNA structural motifs in the hepatitis C viral (HCV) genome that can... more Previously, we described two RNA structural motifs in the hepatitis C viral (HCV) genome that can be processed in vitro by human ribonuclease P (RNase P) enzyme [J. Biol. Chem. 277 (2002) 30606]. One of these structures is located in the internal ribosome entry site and is conserved in the related animal pestiviruses [J. Biol. Chem. 278 (2003) 26844]. Here, we tested two prokaryotic RNase P ribozymes (P RNA) against this conserved structural motif. In vitro experiments indicated that P RNA from Synechocystis sp. can specifically process the viral transcript preparations in a position close to the human RNase P cleavage site. This provides additional support for the presence of an RNA structure similar to tRNA near the AUG start codon and suggests that Synechocystis P RNA may be an active agent for HCV antigenomic interventions.
Biochemical and Biophysical Research Communications, 2004
The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state... more The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA Ser(UCN) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA Ser(UCN) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3 0 processing endonuclease (tRNase Z) confirmed that the efficiency of both 5 0 and 3 0 processing was impaired. A mutant precursor not already processed at the 5 0 end was poorly processed in vitro by tRNase Z. Misprocessing at the 3 0 end further impaired the efficiency and accuracy of 5 0 processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.
Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry sit... more Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5"untranslatable region (5"UTR) of the viral genome. Viral protein synthesis clearly differs from that used by most cellular mRNAs, rendering the IRES an attractive target for novel antiviral compounds. The engineering of RNA compounds is an effective strategy for targeting conserved functional regions in viral RNA genomes. The present work analyses the anti-HCV potential of HH363-24, an in vitro selected molecule composed of a catalytic RNA cleaving domain with an extension at the 3" end that acts as aptamer for the viral 5"UTR. The engineered HH363-24 efficiently cleaved the HCV genome and bound to the essential IIId domain of the IRES region. This action interfered with the proper assembly of the translationally active ribosomal particles 48S and 80S, likely leading to the effective inhibition of IRES function in a hepatic cell line. HH363-24 also efficiently reduced HCV RNA levels up to 70% in a subgenomic replicon system. These findings provide new insights into the development of potential therapeutic strategies based on RNA molecules targeting genomic RNA structural domains and highlight the feasibility of generating novel engineered RNAs as potent antiviral agents.
We have previously described a developmentally regulated mRNA in maize that accumulates in mature... more We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted. RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MAlbencoded protein binds preferentially to uridine-and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.
The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutatio... more The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.
We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum sa... more We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection.
Nietzsche was the first major philosopher contesting Darwin's principle of «struggle for existenc... more Nietzsche was the first major philosopher contesting Darwin's principle of «struggle for existence» as the motor for evolution. Nietzsche opposed to this principle that of «struggle for power». Nietzsche did not deny natural selection, but thought that selection acted essentially on those transformations produced by the struggle for power. The testable hypothesis proposed by Nietzsche is that the victory in the struggle for power does not belong to the strongest, most fortunately constituted individual but rather to the more abundant, meanest individuals (Table ). However, science has not provided a mechanistic formulation of the Nietzschean concept of power relationships capable of undertaking the challenge of some experimental exploration. In this essay we propose that there has been a theoretical formulation, amenable to experimental analysis, which provides a test of power relationships among components of a population of individuals. We refer to the self-replication model of M. Eigen formulated to describe early replicons at the origin of life, and which has found an application in the understanding of RNA viruses. We describe experimental observations on competition among RNA genomes, and we relate the findings to Nietzschean power relationships.
Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies ... more Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previ...
Since January 2020 Elsevier has created a COVID-19 resource centre with free information in Engli... more Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre -including this research content -immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (H... more The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (HRGP) was determined by in situ hybridization. During normal development of roots and leaves, the expression of the gene was transient and particularly high in regions initiating vascular elements and associated sclerenchyma. Its expression was also associated with the differentiation of vascular elements in a variety of other tissues. The gene encoded an HRGP that had been extracted from the cell walls of maize suspension culture cells and several other embryonic and post-embryonic tissues. The gene was present in one or two copies in different varieties of maize and in the related monocots teosinte and sorghum. A single gene was cloned from maize using a previously characterized HRGP cDNA clone . Plant MOI. Biol. 11, 483-4931. In addition to the coding sequences for the HRGP and an N-terminal signal sequence, the gene contained a single intron in the nontranslated 3' end.
Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the... more Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. Picornavirus IRES are organized in structural domains, in which the terminal stem–loops participate in functional RNA–protein interactions. However, the mechanistic role performed by the central domain during internal initiation has not been elucidated yet. Here we show that the foot-and-mouth-disease virus IRES contains a structural motif that serves in vitro as substrate for the Synechocystis sp. RNase P ribozyme, a structure-dependent endonuclease that participates in tRNA precursor processing. Recognition of the IRES substrate was dose dependent, required high magnesium concentration, and resulted in the formation of cleavage products with 5′ phosphate and 3′ hydroxyl ends. Mapping of the core recognition motif indicated that it overlapped with the apical region of the central domain. Two IRES constructs containing nucleotide substitutions...
Previous studies have identified a set of highly phosphorylated proteins of 23-25 kDa accumulated... more Previous studies have identified a set of highly phosphorylated proteins of 23-25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants. During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23-25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABAdeficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos. These results indicate that the 23-25 kDa protein set is a new member of the recently described class of proteins involved in generalized plant ABA responses. The different pattern of expression for the ABA-regulated 23-25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.
In the course of a study conducted from 1992 through 1994 of the efficacy of screening blood dono... more In the course of a study conducted from 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. Methods. Of 222 of the surgeon's patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. Results. The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusionassociated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P Ͻ 0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of a phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. Conclusions. Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery. (
We have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This g... more We have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction, rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vpl mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vpl contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.
Previously, we described two RNA structural motifs in the hepatitis C viral (HCV) genome that can... more Previously, we described two RNA structural motifs in the hepatitis C viral (HCV) genome that can be processed in vitro by human ribonuclease P (RNase P) enzyme [J. Biol. Chem. 277 (2002) 30606]. One of these structures is located in the internal ribosome entry site and is conserved in the related animal pestiviruses [J. Biol. Chem. 278 (2003) 26844]. Here, we tested two prokaryotic RNase P ribozymes (P RNA) against this conserved structural motif. In vitro experiments indicated that P RNA from Synechocystis sp. can specifically process the viral transcript preparations in a position close to the human RNase P cleavage site. This provides additional support for the presence of an RNA structure similar to tRNA near the AUG start codon and suggests that Synechocystis P RNA may be an active agent for HCV antigenomic interventions.
Biochemical and Biophysical Research Communications, 2004
The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state... more The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA Ser(UCN) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA Ser(UCN) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3 0 processing endonuclease (tRNase Z) confirmed that the efficiency of both 5 0 and 3 0 processing was impaired. A mutant precursor not already processed at the 5 0 end was poorly processed in vitro by tRNase Z. Misprocessing at the 3 0 end further impaired the efficiency and accuracy of 5 0 processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.
Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry sit... more Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5"untranslatable region (5"UTR) of the viral genome. Viral protein synthesis clearly differs from that used by most cellular mRNAs, rendering the IRES an attractive target for novel antiviral compounds. The engineering of RNA compounds is an effective strategy for targeting conserved functional regions in viral RNA genomes. The present work analyses the anti-HCV potential of HH363-24, an in vitro selected molecule composed of a catalytic RNA cleaving domain with an extension at the 3" end that acts as aptamer for the viral 5"UTR. The engineered HH363-24 efficiently cleaved the HCV genome and bound to the essential IIId domain of the IRES region. This action interfered with the proper assembly of the translationally active ribosomal particles 48S and 80S, likely leading to the effective inhibition of IRES function in a hepatic cell line. HH363-24 also efficiently reduced HCV RNA levels up to 70% in a subgenomic replicon system. These findings provide new insights into the development of potential therapeutic strategies based on RNA molecules targeting genomic RNA structural domains and highlight the feasibility of generating novel engineered RNAs as potent antiviral agents.
We have previously described a developmentally regulated mRNA in maize that accumulates in mature... more We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted. RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MAlbencoded protein binds preferentially to uridine-and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.
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