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At least 559 records · Page 31Linked to original sources

PubMed2006

Exploring RNA virus replication in Xenopus oocytes.

Microinjection of poliovirus RNA in Xenopus oocytes initiates a complete and authentic viral replication cycle that yields newly synthesized infectious virus. This system can be used to study the molecular mechanism of the different steps involved in virus replication. Interestingly, viral replication only occurs if poliovirus RNA is coinjected with factors present in HeLa extracts. We have determined that two HeLa cell factors are required for viral replication in oocytes, one involved in initiation of translation (polio translation factor) and the other in RNA synthesis. Thus, microinjection in oocytes provides a strategy to identify and further analyze the function of these host cell factors and to biochemically dissect the mechanism of initiation of poliovirus translation and RNA synthesis. Here, we review protocols, approaches, and potential issues that can be addressed using the oocyte system.

Animals↗

PubMed1975

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.

Adenine Nucleotides↗

PubMed2004

The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein.

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.

Antiviral Agents↗

PubMed1997

Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase II.

To develop vector systems that combine high transcription activity with biologically safe delivery vehicles, we have explored the use of RNA replication to amplify mRNAs, by using flock house virus (FHV) as a model system. The FHV RNA replicase is encoded in the larger of the two segments that comprise the viral positive-sense RNA genome. A cDNA copy of this self-replicating RNA was precisely positioned between a promoter site for cellular RNA polymerase II and a cDNA encoding a self-cleaving ribozyme from hepatitis delta virus. Transfection of this plasmid into cultured BHK cells resulted in prolonged, autonomous FHV RNA replication in the cytoplasm and substantial amplification of the RNA replicon. The replicase also amplified RNA transcribed from a second plasmid of similar design that contained a cDNA copy of the other FHV genome segment. These results constitute a significant step toward the harnessing of nodaviral RNA replication as the basis of a versatile vector system.

Animals↗

PubMed1992

Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.

Alanine Transaminase↗

PubMed1990

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA.

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.

Animals↗

PubMed2004

A complex network of RNA-RNA interactions controls subgenomic mRNA transcription in a tombusvirus.

Eukaryotic (+)-strand RNA viruses utilize a wide variety of gene expression strategies to achieve regulated production of their viral proteins. A common mechanism used by many is to transcribe viral subgenomic (sg) mRNAs. Transcription of sg mRNA2 in tombusviruses allows for expression of the p19 suppressor of gene silencing and p22 movement proteins. We have investigated the mechanism of transcription of this sg mRNA in Tomato bushy stunt virus and have determined that this process is facilitated by no less than three different RNA modules that are located throughout the viral genome. These RNA units perform distinct tasks and function via long-distance RNA-RNA interactions. Systematic deconstruction of the RNA network and analysis of related RNA promoter elements allowed us to identify fundamental properties necessary for productive sg mRNA2 transcription. Collectively, our results (i) establish specific roles for the different RNA components of a multipartite RNA-based control system, (ii) support a premature termination mechanism for tombusvirus sg mRNA transcription and (iii) reveal a close mechanistic relationship between sg mRNA transcription, viral RNA replication and RNA recombination.

Base Pairing↗

PubMed2000

In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.

Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0. 0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury.

5' Untranslated Regions↗

PubMed2002

The primary nucleotide sequence of the bovine leukemia virus RNA packaging signal can influence efficient RNA packaging and virus replication.

Two RNA stem-loop structures in the gag gene have been implicated as representing the primary encapsidation (packaging) signal for bovine leukemia virus (BLV), a member of the Delta retrovirus of the Retroviridae. In this study, we conducted an analysis of these RNA structures, stem loop 1 (SL1) and stem loop 2 (SL2), to determine if both the loop and the stem nucleotide bases are important for RNA encapsidation. We have found that the primary sequence of the unpaired bases located in the loop regions of both SL1 and SL2 are important for efficient RNA encapsidation and virus replication. The primary sequence of the bases that form the stems for both SL1 and SL2 was observed to aid in efficient encapsidation and replication. We also observed that the order of SL1 and SL2 is important for RNA encapsidation and virus replication efficiency. A viral RNA with two copies of either SL1 or SL2 was found to replicate and package RNA as efficiently as a viral RNA with only one copy of SL1 or SL2. This provides evidence that SL1 and SL2 are not functionally equivalent. Sequences from human T cell leukemia virus type 1 (HTLV-1) that are located in the same region of HTLV-1 as the SL1 and SL2 of BLV were used to replace the BLV SL1, SL2, or both in a BLV RNA. These BLV RNAs were still encapsidated and replicated, suggesting that these sequences may function as an encapsidation signal in HTLV-1. The chimeric RNAs did not replicate as well as the parental, indicating that the primary nucleotide sequence along with the secondary and tertiary structure of the RNA plays a role in efficient RNA encapsidation and replication.

Animals↗

PubMed1990

Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs.

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

Animals↗

PubMed2001

HCV RNA-dependent RNA polymerase replicates in vitro the 3' terminal region of the minus-strand viral RNA more efficiently than the 3' terminal region of the plus RNA.

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3' end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3' UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3' end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3' end of the minus-strand RNA: (a) the presence of a C residue as the 3' terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3' end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.

Base Sequence↗

PubMed1995

A cis-acting viral protein is not required for the replication of a coronavirus defective-interfering RNA.

Mouse hepatitis virus (MHV), a coronavirus, generates defective-interfering (DI) RNAs of different sizes during passages at high multiplicities of infection. All MHV DI RNAs characterized so far contain an open reading frame (ORF) encoding a fused viral protein; in addition, DI RNAs with a long ORF have a competitive advantage over those with a shorter ORF. These findings suggest that DI RNA replication may require an ORF encoding a cis-acting viral protein. In this study, we used a naturally occurring DI RNA and inserted a 12-nucleotide (nt) amber-mutation linker at various positions to truncate the ORF. Most of the mutants replicated as well as the wild-type DI RNA, irrespective of the presence or absence and the length of the ORF in the RNA. Sequence analysis showed that all of the mutants retained the insertional mutations even after two viral passages in tissue culture, establishing that the mutant DI RNAs replicated. We have further introduced two 3-nucleotide substitutions of the first two AUG codons of the ORF, thus completely closing the ORF. This DI RNA replicated as well as the wild-type DI, but, after a single passage, the majority of the mutant RNAs was replaced by recombinant RNAs which contain a restored functional ORF. However, an additional insertion of a 12-nt amber-mutation linker downstream of the AUG substitutions prevented recombination, and the DI RNA still replicated. These data indicate that DI RNA replication does not require a DI-specific ORF encoding cis-acting viral proteins and that a 12-nucleotide insertion could prevent or delay the occurrence of RNA recombination, suggesting the importance of direct or indirect RNA alignment in homologous RNA recombination.

Animals↗

PubMed2005

Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci.

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP-NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

Animals↗

PubMed2003

Functional analysis of the stem-loop structures at the 5' end of the Aichi virus genome.

Aichi virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5' end) within the 5'-end 120 nucleotides of the genome. We have already shown that the most 5'-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during virus infection.

5' Untranslated Regions↗

PubMed2006

RNA-templated replication of hepatitis delta virus: genomic and antigenomic RNAs associate with different nuclear bodies.

Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to alpha-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both alpha-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was alpha-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was alpha-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.

Amanitins↗

PubMed2006

Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1).

Although replicon systems for hepatitis C virus (HCV) recently developed have enabled the replication of HCV in cultured cells, limited genotypes are available for them. We have isolated HCV cDNA of genotype 2a (JFH-1 strain) from serum of a patient with fulminant hepatitis. A subgenomic replicon of JFH-1 was constructed and compared with the HCV replicon of genotype 1b (Con1 NK5.1) which possessed adaptive mutations. Huh7 cells transfected with replicon RNAs that had been transcribed in vitro were cultured in the presence of neomycin sulfate (G418), and selected colonies were isolated and expanded. Then, growth rates and replication of HCV RNA were evaluated on isolated cells hosting replicons. Saturation densities were lower for cells propagating JFH-1 than Con1 NK5.1 or untransfected Huh7 cells, and the mean doubling time was longer for JFH-1 than for Huh7 cells. Levels of HCV RNA replication in isolated clones were similar between JFH-1 and Con1 NK5.1 cells. Replication of RNA decreased reciprocally with cell densities in both JFH-1 and Con1 NK5.1 cells. The replication of HCV RNA was more resistant to interferon-alpha in JFH-1 than in Con1 NK5.1 cells based on the comparison of an inhibitory concentration of 50%. In conclusion, we found differences between HCV replicon clones of genotypes 1b and 2a. However, these differences may result from strain-specific characteristics, such as the source of HCV, rather than characteristics of distinct genotypes. Therefore, further investigation may be needed on more HCV isolates of diverse genotypes.

Cell Division↗

PubMed1991

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

Acquired Immunodeficiency Syndrome↗

PubMed1984

Structure of poliovirus replicative intermediate RNA. Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative.

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4'-aminomethyl-4,5',8-trimethylpsoralen. After purification from infected cells, undenatured RI appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.

Base Composition↗