CORD-19-Sentences  

CORD-19:003f719a05b7a35ea4b778ded17cae230f5a0343 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-90 Sentence denotes La, PTB, and PAB proteins bind to the 3 0 untranslated region of Norwalk virus genomic RNA
TextSentencer_T1 0-90 Sentence denotes La, PTB, and PAB proteins bind to the 3 0 untranslated region of Norwalk virus genomic RNA
TextSentencer_T2 92-100 Sentence denotes Abstract
TextSentencer_T2 92-100 Sentence denotes Abstract
TextSentencer_T3 101-184 Sentence denotes Noroviruses are human enteric caliciviruses for which no cell culture is available.
TextSentencer_T3 101-184 Sentence denotes Noroviruses are human enteric caliciviruses for which no cell culture is available.
TextSentencer_T4 185-285 Sentence denotes Consequently, the mechanisms and factors involved in their replication have been difficult to study.
TextSentencer_T4 185-285 Sentence denotes Consequently, the mechanisms and factors involved in their replication have been difficult to study.
TextSentencer_T5 286-441 Sentence denotes In an attempt to analyze the cis-and trans-acting factors that could have a role in NV replication, the 3 0 -untranslated region of the genome was studied.
TextSentencer_T5 286-441 Sentence denotes In an attempt to analyze the cis-and trans-acting factors that could have a role in NV replication, the 3 0 -untranslated region of the genome was studied.
TextSentencer_T6 442-541 Sentence denotes Use of Zuker's mfold-2 software predicted that NV 3 0 UTR contains a stem-loop structure of 47 nts.
TextSentencer_T6 442-541 Sentence denotes Use of Zuker's mfold-2 software predicted that NV 3 0 UTR contains a stem-loop structure of 47 nts.
TextSentencer_T7 542-635 Sentence denotes Proteins from HeLa cell extracts, such as La and PTB, form stable complexes with this region.
TextSentencer_T7 542-635 Sentence denotes Proteins from HeLa cell extracts, such as La and PTB, form stable complexes with this region.
TextSentencer_T8 636-821 Sentence denotes The addition of a poly(A) tail (24 nts) to the 3 0 UTR permits the specific binding of the poly(A) binding protein (PABP) present in HeLa cell extracts, as well as the recombinant PABP.
TextSentencer_T8 636-821 Sentence denotes The addition of a poly(A) tail (24 nts) to the 3 0 UTR permits the specific binding of the poly(A) binding protein (PABP) present in HeLa cell extracts, as well as the recombinant PABP.
TextSentencer_T9 822-1009 Sentence denotes Since La, PTB, and PABP are important trans-acting factors required for viral translation and replication, these RNA-protein interactions may play a role in NV replication or translation.
TextSentencer_T9 822-1009 Sentence denotes Since La, PTB, and PABP are important trans-acting factors required for viral translation and replication, these RNA-protein interactions may play a role in NV replication or translation.
TextSentencer_T10 1011-1252 Sentence denotes Norwalk virus (NV) is the prototype strain of human caliciviruses (HuCV), a group of agents causing highly transmissible viral gastroenteritis, which spreads rapidly through families, institutions, and communities (reviewed in [1] [2] [3] ).
TextSentencer_T10 1011-1252 Sentence denotes Norwalk virus (NV) is the prototype strain of human caliciviruses (HuCV), a group of agents causing highly transmissible viral gastroenteritis, which spreads rapidly through families, institutions, and communities (reviewed in [1] [2] [3] ).
TextSentencer_T11 1253-1456 Sentence denotes Noroviruses are difficult to study because they cannot be cultivated in cell culture; however, molecular techniques have been useful to examine the genome organization and function of the viral proteins.
TextSentencer_T11 1253-1456 Sentence denotes Noroviruses are difficult to study because they cannot be cultivated in cell culture; however, molecular techniques have been useful to examine the genome organization and function of the viral proteins.
TextSentencer_T12 1457-1631 Sentence denotes NV has a single stranded positive-polarity RNA genome that contains three open reading frames (ORF), a short 3 0 untranslated region (UTR) of 66 nts, and a poly(A) tail [4] .
TextSentencer_T12 1457-1631 Sentence denotes NV has a single stranded positive-polarity RNA genome that contains three open reading frames (ORF), a short 3 0 untranslated region (UTR) of 66 nts, and a poly(A) tail [4] .
TextSentencer_T13 1632-1683 Sentence denotes The presence of a 5 0 UTR has not been established.
TextSentencer_T13 1632-1683 Sentence denotes The presence of a 5 0 UTR has not been established.
TextSentencer_T14 1684-1849 Sentence denotes Sequence analysis of ORF 1 shows the characteristic motifs of the 2C helicase, 3C cystein protease, and 3D RNA polymerase present in the picornavirus genome [4, 5] .
TextSentencer_T14 1684-1849 Sentence denotes Sequence analysis of ORF 1 shows the characteristic motifs of the 2C helicase, 3C cystein protease, and 3D RNA polymerase present in the picornavirus genome [4, 5] .
TextSentencer_T15 1850-1904 Sentence denotes ORFs 2 and 3 encode the viral capsid proteins [5, 6] .
TextSentencer_T15 1850-1904 Sentence denotes ORFs 2 and 3 encode the viral capsid proteins [5, 6] .
TextSentencer_T16 1905-2175 Sentence denotes The mechanisms and factors involved in NV translation and RNA replication remain unknown; however, as a positive-stranded RNA virus, the genomic RNA has to be translated into the viral non-structural proteins and function as a template for negative-strand RNA synthesis.
TextSentencer_T16 1905-2175 Sentence denotes The mechanisms and factors involved in NV translation and RNA replication remain unknown; however, as a positive-stranded RNA virus, the genomic RNA has to be translated into the viral non-structural proteins and function as a template for negative-strand RNA synthesis.
TextSentencer_T17 2176-2376 Sentence denotes The viral enzyme required for RNA synthesis has to be the viral RNA-dependent RNA polymerase (RdRp) 3D pol that initiates RNA synthesis at the 3 0 end of the genomic and replicative intermediate RNAs.
TextSentencer_T17 2176-2376 Sentence denotes The viral enzyme required for RNA synthesis has to be the viral RNA-dependent RNA polymerase (RdRp) 3D pol that initiates RNA synthesis at the 3 0 end of the genomic and replicative intermediate RNAs.
TextSentencer_T18 2377-2637 Sentence denotes Since viral RdRps are commonly primerdependent enzymes, preinitiation replication complexes require the presence of structured sequences as cis-acting elements to recruit several other trans-acting factors such as viral and cellular proteins [7] [8] [9] [10] .
TextSentencer_T18 2377-2637 Sentence denotes Since viral RdRps are commonly primerdependent enzymes, preinitiation replication complexes require the presence of structured sequences as cis-acting elements to recruit several other trans-acting factors such as viral and cellular proteins [7] [8] [9] [10] .
TextSentencer_T19 2638-2856 Sentence denotes NV and poliovirus (PV) share a high level of homology between their non-structural proteins, and their polyadenylated tails; thus, it is possible that both RNA viruses use similar mechanisms to replicate their genomes.
TextSentencer_T19 2638-2856 Sentence denotes NV and poliovirus (PV) share a high level of homology between their non-structural proteins, and their polyadenylated tails; thus, it is possible that both RNA viruses use similar mechanisms to replicate their genomes.
TextSentencer_T20 2857-3012 Sentence denotes The poly(A) tail increases infectivity [11] and has been considered an important cis-acting element required for negative-strand RNA synthesis of PV [12] .
TextSentencer_T20 2857-3012 Sentence denotes The poly(A) tail increases infectivity [11] and has been considered an important cis-acting element required for negative-strand RNA synthesis of PV [12] .
TextSentencer_T21 3013-3222 Sentence denotes This element is located at the 3 0 end of the genomic RNA, where the initiation of the RNA synthesis takes place and has a critical role in organizing proteins around the start site of the RNA synthesis [12] .
TextSentencer_T21 3013-3222 Sentence denotes This element is located at the 3 0 end of the genomic RNA, where the initiation of the RNA synthesis takes place and has a critical role in organizing proteins around the start site of the RNA synthesis [12] .
TextSentencer_T22 3223-3405 Sentence denotes This sequence is a specific target for the poly(A) binding protein (PABP), a regulatory factor involved in the control of cellular mRNA stability and translation (reviewed in [13] ).
TextSentencer_T22 3223-3405 Sentence denotes This sequence is a specific target for the poly(A) binding protein (PABP), a regulatory factor involved in the control of cellular mRNA stability and translation (reviewed in [13] ).
TextSentencer_T23 3406-3618 Sentence denotes The PABP bound to PV poly(A) tail interacts directly with the 5 0 end via a protein-protein bridge, promoting the circularization of the genomic RNA, which provides several advantages for viral replication [12] .
TextSentencer_T23 3406-3618 Sentence denotes The PABP bound to PV poly(A) tail interacts directly with the 5 0 end via a protein-protein bridge, promoting the circularization of the genomic RNA, which provides several advantages for viral replication [12] .
TextSentencer_T24 3619-3792 Sentence denotes Besides the cis-acting elements, viral RNA replication also depends on some cellular proteins that form part of the ribonucleoprotein (RNP) complexes formed in the 3 0 ends.
TextSentencer_T24 3619-3792 Sentence denotes Besides the cis-acting elements, viral RNA replication also depends on some cellular proteins that form part of the ribonucleoprotein (RNP) complexes formed in the 3 0 ends.
TextSentencer_T25 3793-3977 Sentence denotes It has been reported that La antigen, a nuclear protein involved in RNA polymerase III transcription termination [14] , binds to the 3 0 end of several viral RNAs [15] [16] [17] [18] .
TextSentencer_T25 3793-3977 Sentence denotes It has been reported that La antigen, a nuclear protein involved in RNA polymerase III transcription termination [14] , binds to the 3 0 end of several viral RNAs [15] [16] [17] [18] .
TextSentencer_T26 3978-4140 Sentence denotes The polypyrimidine tract-binding protein (PTB), a 57/60 kDa protein that plays a role in RNA splicing [19] , binds to the 3 0 end of several viral RNAs [20, 21] .
TextSentencer_T26 3978-4140 Sentence denotes The polypyrimidine tract-binding protein (PTB), a 57/60 kDa protein that plays a role in RNA splicing [19] , binds to the 3 0 end of several viral RNAs [20, 21] .
TextSentencer_T27 4141-4341 Sentence denotes Structural and functional similarities in the replication process among polyadenylated RNA viruses suggest that NV may share some of the elements and follow related strategies to replicate its genome.
TextSentencer_T27 4141-4341 Sentence denotes Structural and functional similarities in the replication process among polyadenylated RNA viruses suggest that NV may share some of the elements and follow related strategies to replicate its genome.
TextSentencer_T28 4342-4517 Sentence denotes In order to characterize some elements that could have a role in NV RNA synthesis, the interactions between the 3 0 UTR, the poly(A) tail, and cellular proteins were analyzed.
TextSentencer_T28 4342-4517 Sentence denotes In order to characterize some elements that could have a role in NV RNA synthesis, the interactions between the 3 0 UTR, the poly(A) tail, and cellular proteins were analyzed.
TextSentencer_T29 4518-4696 Sentence denotes The present results indicate that NV 3 0 UTR with a 24 poly(A) tail (3 0 UTR(A)) is able to form a stable stemloop structure of 44 nts as predicted by the mfold-2 software [22] .
TextSentencer_T29 4518-4696 Sentence denotes The present results indicate that NV 3 0 UTR with a 24 poly(A) tail (3 0 UTR(A)) is able to form a stable stemloop structure of 44 nts as predicted by the mfold-2 software [22] .
TextSentencer_T30 4697-4816 Sentence denotes Using a UV-induced crosslinking assay, 10 proteins from HeLa cells were found to bind to the polyadenylated NV 3 0 UTR.
TextSentencer_T30 4697-4816 Sentence denotes Using a UV-induced crosslinking assay, 10 proteins from HeLa cells were found to bind to the polyadenylated NV 3 0 UTR.
TextSentencer_T31 4817-4963 Sentence denotes La from HeLa cells and a recombinant PTB specifically interact with the NV 3 0 UTR (3 0 UTR) while the PABP exclusively binds to the poly(A) tail.
TextSentencer_T31 4817-4963 Sentence denotes La from HeLa cells and a recombinant PTB specifically interact with the NV 3 0 UTR (3 0 UTR) while the PABP exclusively binds to the poly(A) tail.
TextSentencer_T32 4964-4970 Sentence denotes Cells.
TextSentencer_T32 4964-4970 Sentence denotes Cells.
TextSentencer_T33 4971-5147 Sentence denotes HeLa cells were grown in Dulbecco's minimal essential medium supplemented with 10% newborn calf serum, 5000 U penicillin, and 5 lg streptomycin, in a 5% CO 2 incubator at 37°C.
TextSentencer_T33 4971-5147 Sentence denotes HeLa cells were grown in Dulbecco's minimal essential medium supplemented with 10% newborn calf serum, 5000 U penicillin, and 5 lg streptomycin, in a 5% CO 2 incubator at 37°C.
TextSentencer_T34 5148-5230 Sentence denotes The culture medium was changed every other day until the cells reached confluence.
TextSentencer_T34 5148-5230 Sentence denotes The culture medium was changed every other day until the cells reached confluence.
TextSentencer_T35 5231-5292 Sentence denotes Computer prediction of the secondary structure of NV 3 0 UTR.
TextSentencer_T35 5231-5292 Sentence denotes Computer prediction of the secondary structure of NV 3 0 UTR.
TextSentencer_T36 5293-5424 Sentence denotes Computer analysis of the 3 0 UTR and 3 0 UTR(A) of NV was performed using the mfold-2 software [22] in http://www.rpi.edu/ezukerm/.
TextSentencer_T36 5293-5424 Sentence denotes Computer analysis of the 3 0 UTR and 3 0 UTR(A) of NV was performed using the mfold-2 software [22] in http://www.rpi.edu/ezukerm/.
TextSentencer_T37 5425-5471 Sentence denotes In vitro transcription of 3 0 NV genomic RNAs.
TextSentencer_T37 5425-5471 Sentence denotes In vitro transcription of 3 0 NV genomic RNAs.
TextSentencer_T38 5472-5654 Sentence denotes Two RNA molecules were synthesized, both containing nt 7588-7654 of the NV 3 0 UTR genome, (3 0 UTR), and only one of them having a poly(A) tail of approximately 24 nts (3 0 UTR(A)).
TextSentencer_T38 5472-5654 Sentence denotes Two RNA molecules were synthesized, both containing nt 7588-7654 of the NV 3 0 UTR genome, (3 0 UTR), and only one of them having a poly(A) tail of approximately 24 nts (3 0 UTR(A)).
TextSentencer_T39 5655-5795 Sentence denotes The RNAs were produced by in vitro transcription using T7 RNA polymerase from the two PCR-amplified cDNAs containing the respective regions.
TextSentencer_T39 5655-5795 Sentence denotes The RNAs were produced by in vitro transcription using T7 RNA polymerase from the two PCR-amplified cDNAs containing the respective regions.
TextSentencer_T40 5796-5857 Sentence denotes The PCR was performed using the complete NV cDNA as template.
TextSentencer_T40 5796-5857 Sentence denotes The PCR was performed using the complete NV cDNA as template.
TextSentencer_T41 5858-6041 Sentence denotes The sense primer used in the PCR contained the bacteriophage T7 promoter sequence, while the antisense poly(A) primer was used for the addition of the 24 A to the 3 0 UTR PCR product.
TextSentencer_T41 5858-6041 Sentence denotes The sense primer used in the PCR contained the bacteriophage T7 promoter sequence, while the antisense poly(A) primer was used for the addition of the 24 A to the 3 0 UTR PCR product.
TextSentencer_T42 6042-6176 Sentence denotes The PCR was performed for 35 cycles of 94°C for 1 min, 42°C for 1 min, and 75°C for 30 s, using a Perkin-Elmer Cetus DNA thermocycler.
TextSentencer_T42 6042-6176 Sentence denotes The PCR was performed for 35 cycles of 94°C for 1 min, 42°C for 1 min, and 75°C for 30 s, using a Perkin-Elmer Cetus DNA thermocycler.
TextSentencer_T43 6177-6317 Sentence denotes The resulting PCR products were purified by a QIAquiq gel extraction G-50 kit (Qiagen) before they were used as templates for RNA synthesis.
TextSentencer_T43 6177-6317 Sentence denotes The resulting PCR products were purified by a QIAquiq gel extraction G-50 kit (Qiagen) before they were used as templates for RNA synthesis.
TextSentencer_T44 6318-6426 Sentence denotes After transcription reaction, the DNA template was removed by treating the samples with DNase RQ1 (Promega).
TextSentencer_T44 6318-6426 Sentence denotes After transcription reaction, the DNA template was removed by treating the samples with DNase RQ1 (Promega).
TextSentencer_T45 6427-6509 Sentence denotes Unincorporated nucleotides in the reaction mixture were removed by gel filtration.
TextSentencer_T45 6427-6509 Sentence denotes Unincorporated nucleotides in the reaction mixture were removed by gel filtration.
TextSentencer_T46 6510-6636 Sentence denotes For synthesis of radiolabeled RNA transcripts, [a-32 P]UTP or [a-32 P]ATP (Dupont) was included in the transcription reaction.
TextSentencer_T46 6510-6636 Sentence denotes For synthesis of radiolabeled RNA transcripts, [a-32 P]UTP or [a-32 P]ATP (Dupont) was included in the transcription reaction.
TextSentencer_T47 6637-6671 Sentence denotes Preparation of HeLa cell extracts.
TextSentencer_T47 6637-6671 Sentence denotes Preparation of HeLa cell extracts.
TextSentencer_T48 6672-6739 Sentence denotes HeLa cells were prepared using a method previously described [24] .
TextSentencer_T48 6672-6739 Sentence denotes HeLa cells were prepared using a method previously described [24] .
TextSentencer_T49 6740-6777 Sentence denotes Mobility shift electrophoresis assay.
TextSentencer_T49 6740-6777 Sentence denotes Mobility shift electrophoresis assay.
TextSentencer_T50 6778-6871 Sentence denotes Mobility shift electrophoresis assay was performed using a method previously described [23] .
TextSentencer_T50 6778-6871 Sentence denotes Mobility shift electrophoresis assay was performed using a method previously described [23] .
TextSentencer_T51 6872-6992 Sentence denotes The amount of S10 extract from HeLa cells varied from 5 to 20 lg, while 500 nM of the recombinant PABP (rPABP) was used.
TextSentencer_T51 6872-6992 Sentence denotes The amount of S10 extract from HeLa cells varied from 5 to 20 lg, while 500 nM of the recombinant PABP (rPABP) was used.
TextSentencer_T52 6993-7035 Sentence denotes Mobility supershift electrophoresis assay.
TextSentencer_T52 6993-7035 Sentence denotes Mobility supershift electrophoresis assay.
TextSentencer_T53 7036-7156 Sentence denotes As much as 1.5 ll of polyclonal anti-PABP and anti-GADPH antibodies was incubated separately with 10 lg of S10 extracts.
TextSentencer_T53 7036-7156 Sentence denotes As much as 1.5 ll of polyclonal anti-PABP and anti-GADPH antibodies was incubated separately with 10 lg of S10 extracts.
TextSentencer_T54 7157-7263 Sentence denotes The antigen-antibody reaction was allowed to proceed for 30 min on ice before addition of the labeled RNA.
TextSentencer_T54 7157-7263 Sentence denotes The antigen-antibody reaction was allowed to proceed for 30 min on ice before addition of the labeled RNA.
TextSentencer_T55 7264-7348 Sentence denotes The RNA-protein supercomplexes were analyzed in a 6% native gel as described before.
TextSentencer_T55 7264-7348 Sentence denotes The RNA-protein supercomplexes were analyzed in a 6% native gel as described before.
TextSentencer_T56 7349-7389 Sentence denotes UV cross-linking of RNA-protein complex.
TextSentencer_T56 7349-7389 Sentence denotes UV cross-linking of RNA-protein complex.
TextSentencer_T57 7390-7611 Sentence denotes UV-induced crosslinking assay of RNA-protein complexes was performed using a method previously described [24] in the presence of 40 or 60 lg of S10 extract from HeLa cells and 100 or 500 ng of the recombinant PTB protein.
TextSentencer_T57 7390-7611 Sentence denotes UV-induced crosslinking assay of RNA-protein complexes was performed using a method previously described [24] in the presence of 40 or 60 lg of S10 extract from HeLa cells and 100 or 500 ng of the recombinant PTB protein.
TextSentencer_T58 7612-7680 Sentence denotes Immunoprecipitation of UV-induced cross-linked La-protein complexes.
TextSentencer_T58 7612-7680 Sentence denotes Immunoprecipitation of UV-induced cross-linked La-protein complexes.
TextSentencer_T59 7681-7796 Sentence denotes Immunoprecipitation of the cross-linked La-protein complex was performed using a method previously described [23] .
TextSentencer_T59 7681-7796 Sentence denotes Immunoprecipitation of the cross-linked La-protein complex was performed using a method previously described [23] .
TextSentencer_T60 7797-7876 Sentence denotes Five hundred nanograms of monoclonal anti-La or anti-actin antibodies was used.
TextSentencer_T60 7797-7876 Sentence denotes Five hundred nanograms of monoclonal anti-La or anti-actin antibodies was used.
TextSentencer_T61 7877-8001 Sentence denotes The 66 nt long 3 0 UTR from NV was able to form a secondary structure as predicted by the mfold-2 program ( Fig. 1A ) [22] .
TextSentencer_T61 7877-8001 Sentence denotes The 66 nt long 3 0 UTR from NV was able to form a secondary structure as predicted by the mfold-2 program ( Fig. 1A ) [22] .
TextSentencer_T62 8002-8175 Sentence denotes The stem-loop structure formed with the last 47 nts has a DG ¼ À9:6, suggesting its stability and was not altered in the presence of the 24 nts long poly(A) tail (Fig. 1B) .
TextSentencer_T62 8002-8175 Sentence denotes The stem-loop structure formed with the last 47 nts has a DG ¼ À9:6, suggesting its stability and was not altered in the presence of the 24 nts long poly(A) tail (Fig. 1B) .
TextSentencer_T63 8176-8399 Sentence denotes In order to determine if the 3 0 UTR was able to interact with cellular proteins present in HeLa cell extract, mobility shift assays were performed using [a-32 P]UTP labeled RNA representing the complete 3 0 UTR as a probe.
TextSentencer_T63 8176-8399 Sentence denotes In order to determine if the 3 0 UTR was able to interact with cellular proteins present in HeLa cell extract, mobility shift assays were performed using [a-32 P]UTP labeled RNA representing the complete 3 0 UTR as a probe.
TextSentencer_T64 8400-8497 Sentence denotes Under this condition, a major RNA-protein complex was observed as a detectable smear ( Fig. 2A) .
TextSentencer_T64 8400-8497 Sentence denotes Under this condition, a major RNA-protein complex was observed as a detectable smear ( Fig. 2A) .
TextSentencer_T65 8498-8629 Sentence denotes It was also observed that the amount of free RNA decreased when the concentrations of S10 extract increased ( Fig. 2A , lanes 2-5).
TextSentencer_T65 8498-8629 Sentence denotes It was also observed that the amount of free RNA decreased when the concentrations of S10 extract increased ( Fig. 2A , lanes 2-5).
TextSentencer_T66 8630-8797 Sentence denotes Furthermore, when the RNA-protein complex was treated with RNase before electrophoresis through the native gel, two main complexes were observed (Fig. 2B , lanes 2-5).
TextSentencer_T66 8630-8797 Sentence denotes Furthermore, when the RNA-protein complex was treated with RNase before electrophoresis through the native gel, two main complexes were observed (Fig. 2B , lanes 2-5).
TextSentencer_T67 8798-8866 Sentence denotes The major complex, with the fastest migration, was called complex I.
TextSentencer_T67 8798-8866 Sentence denotes The major complex, with the fastest migration, was called complex I.
TextSentencer_T68 8867-9006 Sentence denotes To determine the stability of both RNA-protein complexes, complex formation was performed in the presence of increasing KCl concentrations.
TextSentencer_T68 8867-9006 Sentence denotes To determine the stability of both RNA-protein complexes, complex formation was performed in the presence of increasing KCl concentrations.
TextSentencer_T69 9007-9209 Sentence denotes Both complexes maintained their migration up to 1.2 M of KCl (Fig. 2C , lanes 2-5), which represents more that 10 times the salt concentration under physiological conditions (approximately 120 nM NaCl).
TextSentencer_T69 9007-9209 Sentence denotes Both complexes maintained their migration up to 1.2 M of KCl (Fig. 2C , lanes 2-5), which represents more that 10 times the salt concentration under physiological conditions (approximately 120 nM NaCl).
TextSentencer_T70 9210-9274 Sentence denotes These results suggest that both complexes, I and II, are stable.
TextSentencer_T70 9210-9274 Sentence denotes These results suggest that both complexes, I and II, are stable.
TextSentencer_T71 9275-9606 Sentence denotes The specificity of the RNA-protein binding was further demonstrated in competition experiments (Fig. 2D ), using 10-and 20-fold molar excess of unlabeled homologous or a non-related heterologous (referred to as nts 111-191 from NV that do not interact with cellular proteins as has been described before [23] ) RNAs as competitors.
TextSentencer_T71 9275-9606 Sentence denotes The specificity of the RNA-protein binding was further demonstrated in competition experiments (Fig. 2D ), using 10-and 20-fold molar excess of unlabeled homologous or a non-related heterologous (referred to as nts 111-191 from NV that do not interact with cellular proteins as has been described before [23] ) RNAs as competitors.
TextSentencer_T72 9607-9807 Sentence denotes A 10-fold molar excess of the unlabeled homologous 3 0 UTR strongly reduced complexes I and II formation, while a 20-fold molar excess of the same competitor completely inhibited complex II formation.
TextSentencer_T72 9607-9807 Sentence denotes A 10-fold molar excess of the unlabeled homologous 3 0 UTR strongly reduced complexes I and II formation, while a 20-fold molar excess of the same competitor completely inhibited complex II formation.
TextSentencer_T73 9808-9942 Sentence denotes However, a 20-fold molar excess of unlabeled non-related heterologous RNA transcript was an inefficient competitor (Fig. 2D, lane 4) .
TextSentencer_T73 9808-9942 Sentence denotes However, a 20-fold molar excess of unlabeled non-related heterologous RNA transcript was an inefficient competitor (Fig. 2D, lane 4) .
TextSentencer_T74 9943-10063 Sentence denotes These results strongly suggest that the proteins present in HeLa cell S10 extract bound specifically to the 3 0 UTR RNA.
TextSentencer_T74 9943-10063 Sentence denotes These results strongly suggest that the proteins present in HeLa cell S10 extract bound specifically to the 3 0 UTR RNA.
TextSentencer_T75 10064-10238 Sentence denotes To determine whether the poly(A) tail was also involved in the complex formation, an [a-32 P]UTP labeled 3 0 UTR(A) was incubated with S10 extract from HeLa cells (Fig. 3A) .
TextSentencer_T75 10064-10238 Sentence denotes To determine whether the poly(A) tail was also involved in the complex formation, an [a-32 P]UTP labeled 3 0 UTR(A) was incubated with S10 extract from HeLa cells (Fig. 3A) .
TextSentencer_T76 10239-10384 Sentence denotes Two main complexes, with similar migration patterns to the ones observed with the 3 0 UTR, were detected (Fig. 3A, lanes 3 and 2, respectively) .
TextSentencer_T76 10239-10384 Sentence denotes Two main complexes, with similar migration patterns to the ones observed with the 3 0 UTR, were detected (Fig. 3A, lanes 3 and 2, respectively) .
TextSentencer_T77 10385-10700 Sentence denotes The complexes formed with both RNAs could be different since the poly(A) tail is a target for the binding of cellular proteins; however, RNP complexes were treated with RNAses, thus neither the poly(A) tail nor the complex formed with it, which were not labeled with [a-32 P]UTP, can be detected by autoradiography.
TextSentencer_T77 10385-10700 Sentence denotes The complexes formed with both RNAs could be different since the poly(A) tail is a target for the binding of cellular proteins; however, RNP complexes were treated with RNAses, thus neither the poly(A) tail nor the complex formed with it, which were not labeled with [a-32 P]UTP, can be detected by autoradiography.
TextSentencer_T78 10701-10802 Sentence denotes To visualize the complexes formed with the poly(A) tail, [a-32 P]ATP was used to label both 3 0 RNAs.
TextSentencer_T78 10701-10802 Sentence denotes To visualize the complexes formed with the poly(A) tail, [a-32 P]ATP was used to label both 3 0 RNAs.
TextSentencer_T79 10803-11662 Sentence denotes Under these conditions, we could detect differences in the complex (lane 1) or with 5, 10, 15, and 20 lg of S10 extract from HeLa cells (lanes 2-5, respectively). (B) [a-32 P]UTP labeled 3 0 UTR was incubated without (lane 1) or with 5, 10, 15, and 20 lg of HeLa S10 extract (lanes 2-5, respectively) followed by RNase treatment. (C) [a-32 P]UTP labeled 3 0 UTR RNA was incubated without (lane 1) or with 20 lg S10 extract from HeLa cells (lanes 2-5), in the absence (lanes 2) or, the presence of 0.6, 0.9, and 1.2 M KCl (lanes 3-5, respectively). (D) [a-32 P]UTP labeled 3 0 UTR RNA was incubated without (lane 1) or with 20 lg of S10 extract from HeLa cells (2) (3) (4) (5) in the absence (lane 2) or presence of 10-and 20-fold molar excess of homologous (lanes 3 and 4, respectively) or 20-fold molar excess of non-related heterologous competitor (lane 5).
TextSentencer_T79 10803-11662 Sentence denotes Under these conditions, we could detect differences in the complex (lane 1) or with 5, 10, 15, and 20 lg of S10 extract from HeLa cells (lanes 2-5, respectively). (B) [a-32 P]UTP labeled 3 0 UTR was incubated without (lane 1) or with 5, 10, 15, and 20 lg of HeLa S10 extract (lanes 2-5, respectively) followed by RNase treatment. (C) [a-32 P]UTP labeled 3 0 UTR RNA was incubated without (lane 1) or with 20 lg S10 extract from HeLa cells (lanes 2-5), in the absence (lanes 2) or, the presence of 0.6, 0.9, and 1.2 M KCl (lanes 3-5, respectively). (D) [a-32 P]UTP labeled 3 0 UTR RNA was incubated without (lane 1) or with 20 lg of S10 extract from HeLa cells (2) (3) (4) (5) in the absence (lane 2) or presence of 10-and 20-fold molar excess of homologous (lanes 3 and 4, respectively) or 20-fold molar excess of non-related heterologous competitor (lane 5).
TextSentencer_T80 11663-11774 Sentence denotes Complex formation was assayed by electrophoresis on native polyacrylamide gels and detected by autoradiography.
TextSentencer_T80 11663-11774 Sentence denotes Complex formation was assayed by electrophoresis on native polyacrylamide gels and detected by autoradiography.
TextSentencer_T81 11775-11845 Sentence denotes Mobility of complexes I and II is indicated. formation with both RNAs.
TextSentencer_T81 11775-11845 Sentence denotes Mobility of complexes I and II is indicated. formation with both RNAs.
TextSentencer_T82 11846-12140 Sentence denotes The 3 0 UTR and the 3 0 UTR(A) were able to form five complexes (Fig. 3B , lanes 3 and 2, respectively); complexes I, III, and V formed with the 3 0 UTR(A) had a similar migration pattern to complexes II, III, and V formed with the 3 0 UTR, respectively (Fig. 3B, lanes 2 and 3, respectively) .
TextSentencer_T82 11846-12140 Sentence denotes The 3 0 UTR and the 3 0 UTR(A) were able to form five complexes (Fig. 3B , lanes 3 and 2, respectively); complexes I, III, and V formed with the 3 0 UTR(A) had a similar migration pattern to complexes II, III, and V formed with the 3 0 UTR, respectively (Fig. 3B, lanes 2 and 3, respectively) .
TextSentencer_T83 12141-12332 Sentence denotes However, complex II formed with the 3 0 UTR(A), which together with complex V are the most prominent complexes formed with this RNA, did not correspond to any complex formed with the 3 0 UTR.
TextSentencer_T83 12141-12332 Sentence denotes However, complex II formed with the 3 0 UTR(A), which together with complex V are the most prominent complexes formed with this RNA, did not correspond to any complex formed with the 3 0 UTR.
TextSentencer_T84 12333-12414 Sentence denotes Complexes III and IV were not consistently discernible in subsequent experiments.
TextSentencer_T84 12333-12414 Sentence denotes Complexes III and IV were not consistently discernible in subsequent experiments.
TextSentencer_T85 12415-12515 Sentence denotes Complexes formed with the 3 0 UTR were overexposed with respect to those formed with the 3 0 UTR(A).
TextSentencer_T85 12415-12515 Sentence denotes Complexes formed with the 3 0 UTR were overexposed with respect to those formed with the 3 0 UTR(A).
TextSentencer_T86 12516-12769 Sentence denotes These results suggest that the poly(A) tail may influence the formation of the RNAprotein complexes either because it changes the size and conformation of the RNA and/or because it interacts with some other proteins present in the S10 HeLa cell extract.
TextSentencer_T86 12516-12769 Sentence denotes These results suggest that the poly(A) tail may influence the formation of the RNAprotein complexes either because it changes the size and conformation of the RNA and/or because it interacts with some other proteins present in the S10 HeLa cell extract.
TextSentencer_T87 12770-12954 Sentence denotes To determine if the poly(A) tail was able to interact with some HeLa cell proteins, competition experiments using both unlabeled RNAs and a non-related heterologous RNA were performed.
TextSentencer_T87 12770-12954 Sentence denotes To determine if the poly(A) tail was able to interact with some HeLa cell proteins, competition experiments using both unlabeled RNAs and a non-related heterologous RNA were performed.
TextSentencer_T88 12955-13078 Sentence denotes Unlabeled competitor RNAs were preincubated with cytoplasmic extract before the addition of [a-32 P]ATP labeled 3 0 UTR(A).
TextSentencer_T88 12955-13078 Sentence denotes Unlabeled competitor RNAs were preincubated with cytoplasmic extract before the addition of [a-32 P]ATP labeled 3 0 UTR(A).
TextSentencer_T89 13079-13411 Sentence denotes A 25-fold molar excess of unlabeled homologous RNA resulted in an efficient competition of all complexes (Fig. 4A, lane 3) , while the addition of 25-fold molar excess of the unlabeled 3 0 UTR (heterologous RNA) was able to compete efficiently with all complexes, except complex II, which did not compete at all (Fig. 4A, lane 4) .
TextSentencer_T89 13079-13411 Sentence denotes A 25-fold molar excess of unlabeled homologous RNA resulted in an efficient competition of all complexes (Fig. 4A, lane 3) , while the addition of 25-fold molar excess of the unlabeled 3 0 UTR (heterologous RNA) was able to compete efficiently with all complexes, except complex II, which did not compete at all (Fig. 4A, lane 4) .
TextSentencer_T90 13412-13593 Sentence denotes Moreover, the amount of radioactivity present in complex II was higher than the amount present in complex II formed without competitor RNAs (Fig. 4A, lanes 4 and 1, respectively) .
TextSentencer_T90 13412-13593 Sentence denotes Moreover, the amount of radioactivity present in complex II was higher than the amount present in complex II formed without competitor RNAs (Fig. 4A, lanes 4 and 1, respectively) .
TextSentencer_T91 13594-13695 Sentence denotes These results suggest that complex II contains proteins, which bind specifically to the poly(A) tail.
TextSentencer_T91 13594-13695 Sentence denotes These results suggest that complex II contains proteins, which bind specifically to the poly(A) tail.
TextSentencer_T92 13696-13881 Sentence denotes The addition of a 25-fold molar excess of the unlabeled non-related heterologous RNA was able to compete with complex V, suggesting that this complex is not specific (Fig. 4A, lane 5) .
TextSentencer_T92 13696-13881 Sentence denotes The addition of a 25-fold molar excess of the unlabeled non-related heterologous RNA was able to compete with complex V, suggesting that this complex is not specific (Fig. 4A, lane 5) .
TextSentencer_T93 13882-14032 Sentence denotes Since PABP interacts with the poly(A) tail of several mRNAs, the possibility that complex II contained the PABP was analyzed using a supershift assay.
TextSentencer_T93 13882-14032 Sentence denotes Since PABP interacts with the poly(A) tail of several mRNAs, the possibility that complex II contained the PABP was analyzed using a supershift assay.
TextSentencer_T94 14033-14265 Sentence denotes Incubation with 1.5 ll of polyclonal anti-PABP antibodies resulted in the inhibition of complex II formation (Fig. 4B, lane 3) , while the same amount of anti-GADPH antibodies did not alter any complex formation (Fig. 4B, lane 4) .
TextSentencer_T94 14033-14265 Sentence denotes Incubation with 1.5 ll of polyclonal anti-PABP antibodies resulted in the inhibition of complex II formation (Fig. 4B, lane 3) , while the same amount of anti-GADPH antibodies did not alter any complex formation (Fig. 4B, lane 4) .
TextSentencer_T95 14266-14385 Sentence denotes The absence of complex II in the presence of the anti-PABP antibodies demonstrated that PABP was present in complex II.
TextSentencer_T95 14266-14385 Sentence denotes The absence of complex II in the presence of the anti-PABP antibodies demonstrated that PABP was present in complex II.
TextSentencer_T96 14386-14511 Sentence denotes To corroborate that PABP was able to bind to the 3 0 UTR(A) tail, a mobility shift assay with rPABP was performed (Fig. 4C) .
TextSentencer_T96 14386-14511 Sentence denotes To corroborate that PABP was able to bind to the 3 0 UTR(A) tail, a mobility shift assay with rPABP was performed (Fig. 4C) .
TextSentencer_T97 14512-14799 Sentence denotes Two complexes with different migrations were observed when rPABP bound to the [a-32 P]ATP labeled 3 0 UTR(A) (Fig. 4C, lane 3) , but none of them were formed with [a-32 P]ATP labeled 3 0 UTR (Fig. 4C, lane 2) , indicating that the rPABP was able to specifically bind to the poly(A) tail.
TextSentencer_T97 14512-14799 Sentence denotes Two complexes with different migrations were observed when rPABP bound to the [a-32 P]ATP labeled 3 0 UTR(A) (Fig. 4C, lane 3) , but none of them were formed with [a-32 P]ATP labeled 3 0 UTR (Fig. 4C, lane 2) , indicating that the rPABP was able to specifically bind to the poly(A) tail.
TextSentencer_T98 14800-14980 Sentence denotes It has been reported that PABP can bind to the PV 3 0 UTR with a poly(A) tail containing a minimum of eight nts and that its affinity increases as the poly(A) tail lengthens [12] .
TextSentencer_T98 14800-14980 Sentence denotes It has been reported that PABP can bind to the PV 3 0 UTR with a poly(A) tail containing a minimum of eight nts and that its affinity increases as the poly(A) tail lengthens [12] .
TextSentencer_T99 14981-15103 Sentence denotes The poly(A) tail present in the 3 0 UTR(A) is 24 nt long, therefore more than one PABP molecule can bind to this sequence.
TextSentencer_T99 14981-15103 Sentence denotes The poly(A) tail present in the 3 0 UTR(A) is 24 nt long, therefore more than one PABP molecule can bind to this sequence.
TextSentencer_T100 15104-15196 Sentence denotes The two complexes observed after the RNAse could contain different defined amounts of rPABP.
TextSentencer_T100 15104-15196 Sentence denotes The two complexes observed after the RNAse could contain different defined amounts of rPABP.
TextSentencer_T101 15197-15282 Sentence denotes The expression and purification of the rPABP is described by Herold and Andino [12] .
TextSentencer_T101 15197-15282 Sentence denotes The expression and purification of the rPABP is described by Herold and Andino [12] .
TextSentencer_T102 15283-15428 Sentence denotes Mobility shift assays demonstrated that both the 3 0 UTR and 3 0 UTR(A) RNAs were able to form RNAprotein complexes with some HeLa cell proteins.
TextSentencer_T102 15283-15428 Sentence denotes Mobility shift assays demonstrated that both the 3 0 UTR and 3 0 UTR(A) RNAs were able to form RNAprotein complexes with some HeLa cell proteins.
TextSentencer_T103 15429-15518 Sentence denotes Next, UV-cross-linking experiments were performed in an attempt to identify some of them.
TextSentencer_T103 15429-15518 Sentence denotes Next, UV-cross-linking experiments were performed in an attempt to identify some of them.
TextSentencer_T104 15519-15698 Sentence denotes Ten proteins from HeLa cell extract, with apparent molecular masses of 110, 97, 85, 75, 68, 60, 52, 50, 39, and 33 kDa, bound to both RNAs (Fig. 5A, lanes 2 and 3, respectively) .
TextSentencer_T104 15519-15698 Sentence denotes Ten proteins from HeLa cell extract, with apparent molecular masses of 110, 97, 85, 75, 68, 60, 52, 50, 39, and 33 kDa, bound to both RNAs (Fig. 5A, lanes 2 and 3, respectively) .
TextSentencer_T105 15699-15797 Sentence denotes All the bands observed in this assay were disrupted after proteinase K treatment (data not shown).
TextSentencer_T105 15699-15797 Sentence denotes All the bands observed in this assay were disrupted after proteinase K treatment (data not shown).
TextSentencer_T106 15798-16056 Sentence denotes It was not possible to detect any differences in the proteins crosslinked to both RNAs; however, a higher amount of radioactivity was observed in the 68 kDa protein crosslinked to the 3 0 UTR(A) (7%), compared to the same band crosslinked to the 3 0 UTR RNA.
TextSentencer_T106 15798-16056 Sentence denotes It was not possible to detect any differences in the proteins crosslinked to both RNAs; however, a higher amount of radioactivity was observed in the 68 kDa protein crosslinked to the 3 0 UTR(A) (7%), compared to the same band crosslinked to the 3 0 UTR RNA.
TextSentencer_T107 16057-16175 Sentence denotes The increased amount of radioactivity in the 68-kDa band, could correspond to the PABP that binds to the poly(A) tail.
TextSentencer_T107 16057-16175 Sentence denotes The increased amount of radioactivity in the 68-kDa band, could correspond to the PABP that binds to the poly(A) tail.
TextSentencer_T108 16176-16281 Sentence denotes To examine this possibility, a 50-fold molar excess of the homologous and the heterologous RNAs was used.
TextSentencer_T108 16176-16281 Sentence denotes To examine this possibility, a 50-fold molar excess of the homologous and the heterologous RNAs was used.
TextSentencer_T109 16282-16489 Sentence denotes A strong competition of all the proteins was observed with both RNAs (Fig. 5B, lanes 4 and 3, respectively) ; however, the 68-kDa protein competed only with the homologous unlabeled RNA (Fig. 5B, lane 4) .
TextSentencer_T109 16282-16489 Sentence denotes A strong competition of all the proteins was observed with both RNAs (Fig. 5B, lanes 4 and 3, respectively) ; however, the 68-kDa protein competed only with the homologous unlabeled RNA (Fig. 5B, lane 4) .
TextSentencer_T110 16490-16702 Sentence denotes These results suggest that the presence of the poly(A) tail was responsible for the competition of an extra band of 68-kDa, which co-migrates with the 68 kDa band bound to the [a-32 P] ATP labeled 3 0 UTR(A) RNA.
TextSentencer_T110 16490-16702 Sentence denotes These results suggest that the presence of the poly(A) tail was responsible for the competition of an extra band of 68-kDa, which co-migrates with the 68 kDa band bound to the [a-32 P] ATP labeled 3 0 UTR(A) RNA.
TextSentencer_T111 16703-16778 Sentence denotes The molecular mass of this protein co-relates with the PABP molecular mass.
TextSentencer_T111 16703-16778 Sentence denotes The molecular mass of this protein co-relates with the PABP molecular mass.
TextSentencer_T112 16779-16873 Sentence denotes No competition was observed when the non-related heterologous RNA was used (Fig. 5B, lane 5) .
TextSentencer_T112 16779-16873 Sentence denotes No competition was observed when the non-related heterologous RNA was used (Fig. 5B, lane 5) .
TextSentencer_T113 16874-17064 Sentence denotes Some of the proteins that crosslinked to both 3 0 UTR and 3 0 UTR(A) RNAs have the same molecular weight as a number of factors that interact with the 5 0 -and 3 0 UTRs of other RNA viruses.
TextSentencer_T113 16874-17064 Sentence denotes Some of the proteins that crosslinked to both 3 0 UTR and 3 0 UTR(A) RNAs have the same molecular weight as a number of factors that interact with the 5 0 -and 3 0 UTRs of other RNA viruses.
TextSentencer_T114 17065-17193 Sentence denotes Specifically, the proteins with molecular masses of 60 and 52-kDa co-relate with the cellular proteins PTB and La, respectively.
TextSentencer_T114 17065-17193 Sentence denotes Specifically, the proteins with molecular masses of 60 and 52-kDa co-relate with the cellular proteins PTB and La, respectively.
TextSentencer_T115 17194-17362 Sentence denotes To investigate if La protein could be one of the proteins that crosslinked to the 3 0 UTR, an immunoprecipitation after the UV-induced crosslinking assay was performed.
TextSentencer_T115 17194-17362 Sentence denotes To investigate if La protein could be one of the proteins that crosslinked to the 3 0 UTR, an immunoprecipitation after the UV-induced crosslinking assay was performed.
TextSentencer_T116 17363-17582 Sentence denotes A labeled 52 kDa protein, crosslinked to the 3 0 UTR, was immunoprecipitated by anti-La antibodies (Fig. 5C, lane 3) , while anti-actin antibodies were unable to immunoprecipitate any labeled protein (Fig. 5C, lane 2) .
TextSentencer_T116 17363-17582 Sentence denotes A labeled 52 kDa protein, crosslinked to the 3 0 UTR, was immunoprecipitated by anti-La antibodies (Fig. 5C, lane 3) , while anti-actin antibodies were unable to immunoprecipitate any labeled protein (Fig. 5C, lane 2) .
TextSentencer_T117 17583-17677 Sentence denotes This fact demonstrated that La was one of the proteins that interacted with the 3 0 UTR of NV.
TextSentencer_T117 17583-17677 Sentence denotes This fact demonstrated that La was one of the proteins that interacted with the 3 0 UTR of NV.
TextSentencer_T118 17678-17918 Sentence denotes To analyze the possible interaction of PTB with the NV 3 0 UTR, labeled 3 0 UTR was incubated with a recombinant PTB (rPTB) protein (Fig. 5D) . rPTB was able to bind to the 3 0 UTR at 100 and 500 ng (Fig. 5D, lanes 2 and 3, respectively) .
TextSentencer_T118 17678-17918 Sentence denotes To analyze the possible interaction of PTB with the NV 3 0 UTR, labeled 3 0 UTR was incubated with a recombinant PTB (rPTB) protein (Fig. 5D) . rPTB was able to bind to the 3 0 UTR at 100 and 500 ng (Fig. 5D, lanes 2 and 3, respectively) .
TextSentencer_T119 17919-18133 Sentence denotes However, this rPTB showed a migration slightly different from that of the 57/60 kDa protein from HeLa cell extracts crosslinked to the same RNA (Fig. 5D, lanes 1, and 2-3 , respectively) as 2 and 3, respectively) .
TextSentencer_T119 17919-18133 Sentence denotes However, this rPTB showed a migration slightly different from that of the 57/60 kDa protein from HeLa cell extracts crosslinked to the same RNA (Fig. 5D, lanes 1, and 2-3 , respectively) as 2 and 3, respectively) .
TextSentencer_T120 18134-18203 Sentence denotes After UV-cross-linking, the reaction was followed by RNase treatment.
TextSentencer_T120 18134-18203 Sentence denotes After UV-cross-linking, the reaction was followed by RNase treatment.
TextSentencer_T121 18204-18302 Sentence denotes Crosslinked proteins were loaded on an SDS-10% polyacrylamide gel and detected by autoradiography.
TextSentencer_T121 18204-18302 Sentence denotes Crosslinked proteins were loaded on an SDS-10% polyacrylamide gel and detected by autoradiography.
TextSentencer_T122 18303-18417 Sentence denotes The migration of the UV-crosslinked 68 kDa, rPTB, and the immunoprecipitated La proteins is indicated by an arrow.
TextSentencer_T122 18303-18417 Sentence denotes The migration of the UV-crosslinked 68 kDa, rPTB, and the immunoprecipitated La proteins is indicated by an arrow.
TextSentencer_T123 18418-18445 Sentence denotes previously described [23] .
TextSentencer_T123 18418-18445 Sentence denotes previously described [23] .
TextSentencer_T124 18446-18568 Sentence denotes These results strongly suggest that the 3 0 UTR contains structural elements that permit the interaction with La and rPTB.
TextSentencer_T124 18446-18568 Sentence denotes These results strongly suggest that the 3 0 UTR contains structural elements that permit the interaction with La and rPTB.
TextSentencer_T125 18569-18833 Sentence denotes Most of the mechanisms and factors involved in NV translation and replication remain unknown; however, as a positive-stranded RNA virus, the viral genome must function as a mRNA for viral protein synthesis and as a template for viral negative-strand RNA synthesis.
TextSentencer_T125 18569-18833 Sentence denotes Most of the mechanisms and factors involved in NV translation and replication remain unknown; however, as a positive-stranded RNA virus, the viral genome must function as a mRNA for viral protein synthesis and as a template for viral negative-strand RNA synthesis.
TextSentencer_T126 18834-19284 Sentence denotes The initiation of the negative-strand RNA synthesis takes place at the 3 0 end of the positive polarity RNA and requires the presence of several elements, such as structured sequences (that often consist of multiple discontinuous sequences of the viral RNA, like the 3 0 and 5 0 UTRs and/or coding regions of the RNA molecule) that serve as cis-acting elements or targets for the interaction of several viral and cellular factors [7, 8, 15, 25, 26] .
TextSentencer_T126 18834-19284 Sentence denotes The initiation of the negative-strand RNA synthesis takes place at the 3 0 end of the positive polarity RNA and requires the presence of several elements, such as structured sequences (that often consist of multiple discontinuous sequences of the viral RNA, like the 3 0 and 5 0 UTRs and/or coding regions of the RNA molecule) that serve as cis-acting elements or targets for the interaction of several viral and cellular factors [7, 8, 15, 25, 26] .
TextSentencer_T127 19285-19378 Sentence denotes These RNA-protein complexes have been involved in both viral translation and RNA replication.
TextSentencer_T127 19285-19378 Sentence denotes These RNA-protein complexes have been involved in both viral translation and RNA replication.
TextSentencer_T128 19379-19540 Sentence denotes In the replication process, the presence of cellular proteins could help to recruit and stabilize the RdRp on the initiation sites for viral RNA synthesis [15] .
TextSentencer_T128 19379-19540 Sentence denotes In the replication process, the presence of cellular proteins could help to recruit and stabilize the RdRp on the initiation sites for viral RNA synthesis [15] .
TextSentencer_T129 19541-19687 Sentence denotes In polyadenylated viruses, such as picornavirus and coronavirus, the poly(A) tail is a specific target sequence for the interaction with the PABP.
TextSentencer_T129 19541-19687 Sentence denotes In polyadenylated viruses, such as picornavirus and coronavirus, the poly(A) tail is a specific target sequence for the interaction with the PABP.
TextSentencer_T130 19688-19945 Sentence denotes This poly(A) sequence has been considered one of the major determinants of initiation for negative strand RNA synthesis with a specific role in organizing proteins around the start site and mediating circularization of the RNA molecule [12, [26] [27] [28] .
TextSentencer_T130 19688-19945 Sentence denotes This poly(A) sequence has been considered one of the major determinants of initiation for negative strand RNA synthesis with a specific role in organizing proteins around the start site and mediating circularization of the RNA molecule [12, [26] [27] [28] .
TextSentencer_T131 19946-20110 Sentence denotes Genome circularization has a critical role in the coordination of translation and RNA synthesis and in the location of the RdRp at the appropriate start site [12] .
TextSentencer_T131 19946-20110 Sentence denotes Genome circularization has a critical role in the coordination of translation and RNA synthesis and in the location of the RdRp at the appropriate start site [12] .
TextSentencer_T132 20111-20265 Sentence denotes In this study, evidence that the NV polyadenylated 3 0 UTR contains the sequences required for the interaction with various cellular proteins is provided.
TextSentencer_T132 20111-20265 Sentence denotes In this study, evidence that the NV polyadenylated 3 0 UTR contains the sequences required for the interaction with various cellular proteins is provided.
TextSentencer_T133 20266-20430 Sentence denotes Using the mfold-2 software, it was possible to predict that the 66 nts present in the complete NV 3 0 UTR contained a stem-loop structure formed in the last 47 nts.
TextSentencer_T133 20266-20430 Sentence denotes Using the mfold-2 software, it was possible to predict that the 66 nts present in the complete NV 3 0 UTR contained a stem-loop structure formed in the last 47 nts.
TextSentencer_T134 20431-20562 Sentence denotes It was demonstrated that in the absence of RNAse, the 3 0 UTR was able to form a large RNA-protein complex with HeLa cell proteins.
TextSentencer_T134 20431-20562 Sentence denotes It was demonstrated that in the absence of RNAse, the 3 0 UTR was able to form a large RNA-protein complex with HeLa cell proteins.
TextSentencer_T135 20563-20653 Sentence denotes When RNP complexes were treated with RNases, two RNA-protein complexes were distinguished.
TextSentencer_T135 20563-20653 Sentence denotes When RNP complexes were treated with RNases, two RNA-protein complexes were distinguished.
TextSentencer_T136 20654-20831 Sentence denotes The complete 3 0 UTR was able to form stable RNA-protein complexes with cellular factors from HeLa S10 extract since they were not disrupted by increasing concentrations of KCl.
TextSentencer_T136 20654-20831 Sentence denotes The complete 3 0 UTR was able to form stable RNA-protein complexes with cellular factors from HeLa S10 extract since they were not disrupted by increasing concentrations of KCl.
TextSentencer_T137 20832-20987 Sentence denotes The specificity of the complex formation was analyzed in competition experiments performed in the presence of an excess of homologous or heterologous RNAs.
TextSentencer_T137 20832-20987 Sentence denotes The specificity of the complex formation was analyzed in competition experiments performed in the presence of an excess of homologous or heterologous RNAs.
TextSentencer_T138 20988-21270 Sentence denotes A total reduction of complex II and a significant reduction of complex I formed with the 3 0 UTR and HeLa cell S10 extract were observed when samples were incubated in the presence of homologous, but not heterologous competitor, strongly suggesting that both complexes are specific.
TextSentencer_T138 20988-21270 Sentence denotes A total reduction of complex II and a significant reduction of complex I formed with the 3 0 UTR and HeLa cell S10 extract were observed when samples were incubated in the presence of homologous, but not heterologous competitor, strongly suggesting that both complexes are specific.
TextSentencer_T139 21271-21573 Sentence denotes Since the poly(A) tail is an important element for organizing proteins around the start site of negative strand RNA synthesis [12] , and it is the binding site for the PABP, we wanted to find out if the presence of the poly(A) tail added to the 3 0 UTR modified its interaction with HeLa cell proteins.
TextSentencer_T139 21271-21573 Sentence denotes Since the poly(A) tail is an important element for organizing proteins around the start site of negative strand RNA synthesis [12] , and it is the binding site for the PABP, we wanted to find out if the presence of the poly(A) tail added to the 3 0 UTR modified its interaction with HeLa cell proteins.
TextSentencer_T140 21574-21759 Sentence denotes We could not detect any difference in the RNP complexes formed with the [a-32 P]UTP 3 0 UTR or 3 0 UTR(A), even though the poly(A) tail is a target for the binding of cellular proteins.
TextSentencer_T140 21574-21759 Sentence denotes We could not detect any difference in the RNP complexes formed with the [a-32 P]UTP 3 0 UTR or 3 0 UTR(A), even though the poly(A) tail is a target for the binding of cellular proteins.
TextSentencer_T141 21760-22017 Sentence denotes This finding could be the result of the RNAse treatment, where the poly(A) tail, which was not labeled with the [a-32 P]UTP, alone or bound to cell proteins, could be separated from the labeled complexes and consequently was not detected by autoradiography.
TextSentencer_T141 21760-22017 Sentence denotes This finding could be the result of the RNAse treatment, where the poly(A) tail, which was not labeled with the [a-32 P]UTP, alone or bound to cell proteins, could be separated from the labeled complexes and consequently was not detected by autoradiography.
TextSentencer_T142 22018-22138 Sentence denotes To visualize the complexes formed with the poly(A) tail, [a-32 P]ATP was used to label both 3 0 UTR and 3 0 UTR(A) RNAs.
TextSentencer_T142 22018-22138 Sentence denotes To visualize the complexes formed with the poly(A) tail, [a-32 P]ATP was used to label both 3 0 UTR and 3 0 UTR(A) RNAs.
TextSentencer_T143 22139-22370 Sentence denotes Under this condition, we detected the formation of five complexes with both RNAs, some of them with the same migration patterns, but complex II, formed with the 3 0 UTR(A), did not correspond to any complex formed with the 3 0 UTR.
TextSentencer_T143 22139-22370 Sentence denotes Under this condition, we detected the formation of five complexes with both RNAs, some of them with the same migration patterns, but complex II, formed with the 3 0 UTR(A), did not correspond to any complex formed with the 3 0 UTR.
TextSentencer_T144 22371-22470 Sentence denotes Then complex II could be formed by the poly(A) tail bound to proteins present in HeLa cell extract.
TextSentencer_T144 22371-22470 Sentence denotes Then complex II could be formed by the poly(A) tail bound to proteins present in HeLa cell extract.
TextSentencer_T145 22471-22635 Sentence denotes Since the poly(A) tract is the target site for the PABP interaction, competition experiments of complexes formed with [a-32 P]ATP labeled 3 0 UTR(A) were performed.
TextSentencer_T145 22471-22635 Sentence denotes Since the poly(A) tract is the target site for the PABP interaction, competition experiments of complexes formed with [a-32 P]ATP labeled 3 0 UTR(A) were performed.
TextSentencer_T146 22636-22793 Sentence denotes We could demonstrate that complex II contains proteins that specifically bind to the poly(A) tail, since this complex was not completed with the 3 0 UTR RNA.
TextSentencer_T146 22636-22793 Sentence denotes We could demonstrate that complex II contains proteins that specifically bind to the poly(A) tail, since this complex was not completed with the 3 0 UTR RNA.
TextSentencer_T147 22794-22918 Sentence denotes Moreover, the presence of the PABP in complex II was confirmed in mobility gel supershift assays using anti-PABP antibodies.
TextSentencer_T147 22794-22918 Sentence denotes Moreover, the presence of the PABP in complex II was confirmed in mobility gel supershift assays using anti-PABP antibodies.
TextSentencer_T148 22919-23038 Sentence denotes In addition, an rPABP was also able to specifically bind to the 3 0 UTR(A) RNA; but it did not bind to the 3 0 UTR RNA.
TextSentencer_T148 22919-23038 Sentence denotes In addition, an rPABP was also able to specifically bind to the 3 0 UTR(A) RNA; but it did not bind to the 3 0 UTR RNA.
TextSentencer_T149 23039-23230 Sentence denotes The two bands in the mobility shift assay can be explained due to the presence of the 24 nts long tail that could permit the formation of a RNP complex with more than one PABP molecule [12] .
TextSentencer_T149 23039-23230 Sentence denotes The two bands in the mobility shift assay can be explained due to the presence of the 24 nts long tail that could permit the formation of a RNP complex with more than one PABP molecule [12] .
TextSentencer_T150 23231-23337 Sentence denotes Then RNAse treatment could generate two different complexes containing different defined amounts of rPABP.
TextSentencer_T150 23231-23337 Sentence denotes Then RNAse treatment could generate two different complexes containing different defined amounts of rPABP.
TextSentencer_T151 23338-23452 Sentence denotes Using crosslinking assays, 10 proteins from 110 to 33 kDa bound to both 3 0 UTR and 3 0 UTR(A) RNAs were detected.
TextSentencer_T151 23338-23452 Sentence denotes Using crosslinking assays, 10 proteins from 110 to 33 kDa bound to both 3 0 UTR and 3 0 UTR(A) RNAs were detected.
TextSentencer_T152 23453-23567 Sentence denotes However, no difference between both patterns at the usual electrophoretic migration of PABP (68-72 kDa) was found.
TextSentencer_T152 23453-23567 Sentence denotes However, no difference between both patterns at the usual electrophoretic migration of PABP (68-72 kDa) was found.
TextSentencer_T153 23568-23976 Sentence denotes Even so, the 68 kDa band that crosslinks with the NV3 0 UTR(A) contains more that one protein, since the competition experiments using an excess of homologous RNA competed completely with this band; while the heterologous RNA, which does not contain the poly(A) tail, only competed partially, suggesting that the 68 kDa band contains at least one protein that specifically binds to the poly(A) tail sequence.
TextSentencer_T153 23568-23976 Sentence denotes Even so, the 68 kDa band that crosslinks with the NV3 0 UTR(A) contains more that one protein, since the competition experiments using an excess of homologous RNA competed completely with this band; while the heterologous RNA, which does not contain the poly(A) tail, only competed partially, suggesting that the 68 kDa band contains at least one protein that specifically binds to the poly(A) tail sequence.
TextSentencer_T154 23977-24083 Sentence denotes Experiments to identify the 97 and the 68-kDa protein (other than the PAPB) are currently being performed.
TextSentencer_T154 23977-24083 Sentence denotes Experiments to identify the 97 and the 68-kDa protein (other than the PAPB) are currently being performed.
TextSentencer_T155 24084-24256 Sentence denotes Besides the finding of PABP bound to the 3 0 UTR(A), two other cellular proteins, La and rPTB, were identified by immunoprecipitation and crosslinking assays, respectively.
TextSentencer_T155 24084-24256 Sentence denotes Besides the finding of PABP bound to the 3 0 UTR(A), two other cellular proteins, La and rPTB, were identified by immunoprecipitation and crosslinking assays, respectively.
TextSentencer_T156 24257-24354 Sentence denotes This is an interesting finding because both proteins are also associated to some viral functions:
TextSentencer_T156 24257-24354 Sentence denotes This is an interesting finding because both proteins are also associated to some viral functions:
TextSentencer_T157 24355-24522 Sentence denotes La has an essential role in PV translation [29] while PTB is necessary for PV and hepatitis C (HCV) translation [16, 30] and mouse hepatitis virus RNA synthesis [31] .
TextSentencer_T157 24355-24522 Sentence denotes La has an essential role in PV translation [29] while PTB is necessary for PV and hepatitis C (HCV) translation [16, 30] and mouse hepatitis virus RNA synthesis [31] .
TextSentencer_T158 24523-24975 Sentence denotes Even though there is no evidence about the mechanisms that operate in NV replication, the work reported here provides new data pointing towards the identification of some of the elements that could have a role in these processes, such as the presence of a stem-loop structure within the polyadenylated 3 0 UTR of NV genomic RNA, and the identification of some of the cellular proteins that specifically bind to this region, including La, PTB, and PABP.
TextSentencer_T158 24523-24975 Sentence denotes Even though there is no evidence about the mechanisms that operate in NV replication, the work reported here provides new data pointing towards the identification of some of the elements that could have a role in these processes, such as the presence of a stem-loop structure within the polyadenylated 3 0 UTR of NV genomic RNA, and the identification of some of the cellular proteins that specifically bind to this region, including La, PTB, and PABP.
TextSentencer_T159 24976-25221 Sentence denotes The identification of these cis-and trans-elements, considered molecules that have essential roles in translation and replication of other positive-sense RNA viruses, can help understand the specific mechanisms that take place in NV replication.
TextSentencer_T159 24976-25221 Sentence denotes The identification of these cis-and trans-elements, considered molecules that have essential roles in translation and replication of other positive-sense RNA viruses, can help understand the specific mechanisms that take place in NV replication.
TextSentencer_T160 25222-25395 Sentence denotes Further analyses, including functional studies, are currently being performed to assess the elements and strategies followed by NV to translate and replicate its RNA genome.
TextSentencer_T160 25222-25395 Sentence denotes Further analyses, including functional studies, are currently being performed to assess the elements and strategies followed by NV to translate and replicate its RNA genome.