Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture.

Improved semiquantitative Western blot technique with increased quantification range.

With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays.

Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans.

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5' and 3' untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.

Hepatitis C virus variability: sequence analysis of an isolate after 10 years of chronic infection.

Hepatitis C virus (HCV) variability was analyzed based upon an isolate which had caused the infection of more than 2500 women in 1978/79. Genome consensus sequences of two isolates obtained from the infectious source (HCV-AD78) and from a chronic hepatitis patient 10 years after the acute infection were determined. The entire open reading frame (ORF) exhibited 3.2 x 10(-3) nucleotide substitutions per site per year (deltant). Core (0.7 x 10(-3) deltant) and NS5B (1.9 x 10(-3) deltant) were found to be most conserved genes, while E2 (4.7 x 10(-3) deltant) with hypervariable region 1 (HVR1) (23 x 10(-3) deltant) was the most variable followed by p7 (4.2 x 10(-3) deltant). In the entire ORF transitions were 4.5 times more frequent than transversions while for the HVR1 this bias was turned. As an indicator of relative selective pressure on the proteins the rates of nonsynonymous to synonymous substitutions (dN/dS) were determined. The obtained values exceeded 1.0 only for E2 (dN/dS = 1.3). A subdivision of the entire ORF into 88 overlapping sections, each containing 300 nucleotides, led to a more precise analysis of HCV diversity. Besides for E2 an increased variability was mainly detected for three other regions: (a) the C terminal neighbouring region of E2 including p7, (b) the genome fragment extending from approximately the middle of NS3 to NS4B, and (c) the segment corresponding to the C-terminus of the NS5A protein. The variable region in NS5A was situated carboxyterminal to the predicted interferon sensitivity determining region (ISDR). These results suggest which regions other than HVR1 might contribute to persistence of the virus by the mechanism of immunescape.

A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA.

Bovine viral diarrhea virus (BVDV), a Pestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5' and 3' ends. The 5' UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5'-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5' end of the viral genome (hairpin Ia) as well as at the 3' end of the complementary negative-strand replication intermediate [termed hairpin Ia (-)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5'-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (-) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (-) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5'-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.

Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study.

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products-negative-strand intermediate and progeny positive-strand RNA-in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein-in particular the ATPase/RNA helicase-play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.

Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication.

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.

Characterization of an autonomous subgenomic pestivirus RNA replicon.

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+)-stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules. An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.

Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9.

In the assembly of a prespliceosome, U2 small nuclear ribonucleoprotein (snRNP) functions in pre-messenger RNA (mRNA) splicing together with splicing factors (SFs) 3a, SF3b, and several other proteins. The 17S but not the 12S form of U2 snRNP is active in splicing-complex formation. Here it is shown that the SF3a subunits correspond to three of the 17S U2 snRNP-specific polypeptides. SF3a interacts with U2 snRNP in the presence of SF3b to generate a structure similar to 17S U2 snRNP, which suggests a function for SF3a and SF3b in the incorporation of U2 snRNP into the spliceosome. Furthermore, the 60-kilodalton subunit of SF3a is related to the yeast splicing protein PRP9.

Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes.

Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form. By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.

Interaction of the human autoantigen p150 with splicing snRNPs.

An important goal of studies on pre-mRNA splicing is to identify factors that mediate the snRNP-snRNP and snRNP-pre-mRNA interactions that take place in the spliceosome. The U4/U6 snRNP is one of the four snRNPs that are subunits of spliceosomes. A rare patient autoimmune serum (MaS serum) has recently been identified that specifically immunoprecipitates U4/U6 snRNP from HeLa cell extracts through recognition of a 150 kDa autoantigen (p150) (Okano and Medsger, Journal of Immunology, 146, 535-542, 1991). Here we show that in addition to U4/U6 snRNP, p150 can also be detected associated with 20 S U5, U4/U6.U5 and 17 S U2 snRNPs, but not with U1 snRNP. In each particle p150 is present in sub-stoichiometric levels relative to the major snRNP proteins. We show that MaS serum selectively immunoprecipitates a sub-population of U4/U6 snRNPs in which the m3G-cap structure is masked and that p150 is preferentially associated with U6 snRNA in the U4/U6 particle. Anti-p150 antibodies show widespread nucleoplasmic staining, excluding nucleoli, with an elevated concentration in coiled bodies. This changes to a discrete punctate pattern when cells are treated with alpha-amanitin. Both the cytological and biochemical data indicate that the p150 autoantigen is a snRNP-associated factor in vivo. We also present biochemical evidence confirming that assembly of U4/U6 and U5 snRNPs into a U4/U6.U5 tri-snRNP particle is an integral step in the spliceosome assembly pathway. Addition of the purified U4/U6.U5 tri-snRNP restores splicing activity to inactivated HeLa nuclear extracts in which splicing had been inhibited by specific depletion of either the U4/U6 or U5 snRNPs.

Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.

A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins.

One of the consequences of the heat shock response is a shutdown of pre-mRNA splicing, a phenomenon that can be reproduced in extracts prepared from heat-shocked cells. The block in splicing occurs before the covalent modifications that generate spliced mRNA at the level of spliceosome formation. We have used extracts prepared from heat-shocked cells as a complementation system to characterize and partially purify a protein factor that is inactivated during the in vivo heat shock. The activity functions in the formation of the active spliceosome by assembling U4/U6 and U5 snRNPs into a triple snRNP particle. The factor appears to be different from previously isolated splicing factors and is functionally equivalent to several polypeptides that are specifically associated with the purified triple snRNP but not with individual U4/U6 or U5 snRNPs. Our data confirm the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a triple snRNP complex and show for the first time a function of specific snRNP-associated polypeptides in the mammalian splicing pathway.

Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells.

We describe the isolation and biochemical characterization of [U4/U6.U5] tri-snRNP complexes from HeLa cells under nondenaturing conditions using a monoclonal antibody reacting with the U5-specific 100-kD protein. We show that the [U4/U6.U5] complex contains five previously unobserved proteins with molecular masses of 90, 60, 27, 20, and 15.5 kD, in addition to the core proteins, common to the U4/U6, U5, U1, and U2 snRNPs, and the U5-specific proteins, as found in 20S U5 snRNPs. With approximately 20 distinct snRNP proteins the complexity of the [U4/U6.U5] tri-snRNP is surprising. One or more of the five proteins found exclusively in the 25S [U4/U6.U5] tri-snRNP appears to be involved in the assembly of the tri-snRNP complex, as, in an in vitro reconstitution assay, purified 20S U5 and 10S U4/U6 snRNPs formed stable 25S [U4/U6.U5] complexes only in the presence of the free tri-snRNP-specific proteins. The formation of the [U4/U6.U5] complex in vitro does not require ATP, and the stability of the purified tri-snRNP complex is not affected by ATP to a measurable extent. However, the native [U4/U6.U5] displays a kinase activity that is absent in isolated U5: A 52-kD protein present in both U5 and [U4/U6.U5] is phosphorylated only in the latter. The function of this phosphorylation is unclear thus far; it may be involved in the activation of [U4/U6.U5] in the spliceosome.

Isolation and characterization of Bacillus stearothermophilus 30S and 50S ribosomal protein mutations.

Bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated. Many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system. This collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.

Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events.

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains. The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions. It is compatible with the previously observed effects of box A in antitermination.