Recombinant RNA Research Articles

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

The approximately 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains approximately 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.

Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 microM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.

Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.

RNA Structural Rearrangement via Unwinding and Annealing by the Cyanobacterial RNA Helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.

Indeterminate third-generation hepatitis C recombinant immunoblot assay and HCV RNA analysis: Isolated reactivity against NS5 associated with HCV viraemia in clinical patients but not blood donors

It has previously been reported that singular reactivity against NS5 in the third generation hepatitis C virus (HCV) confirmatory recombinant immunoblot assay (RIBA-3) is not associated with detectable HCV RNA in sera. In order to investigate the significance of indeterminate HCV RIBA-3 results with particular regard to NS5, 165 sera with indeterminate RIBA-3 results were analysed for the presence of HCV RNA. Sera from blood donors constituted 58 of the 165 samples, whereas 11 were from immunocompromized patients. The remaining 96 sera were from non-immunosuppressed clinical patients. Of the 107 RIBA-3 indeterminate samples from clinical patients, 19 were HCV RNA positive (18%), with 3 of the 57 (5%) NS5 reactive samples having detectable HCV RNA. None of the 58 indeterminate samples obtained from blood donors had detectable HCV RNA. Thus, having an indeterminate RIBA-3, including singular reactivity against NS5, may be associated with the presence of detectable levels of HCV RNA in clinical patients but not necessarily in blood donors.

Identification ofrpoBCGenes Encoding for β and β′ Subunits of RNA Polymerase in a Deep-Sea Piezophilic Bacterium,Shewanella violaceaStrain DSS12

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was sigma(70) containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the beta and beta' subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni(2+) affinity column. In vitro reconstitution using all proteins for the holoenzyme (alpha, beta, beta', sigma(70)) was carried out and the activity of the recombinant RNA polymerase was detected.

The Effect of Telomerase Antisense on the Differentiation of CD34+ Cells from Bone Marrow to Blood Cells.

Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense (TA) expression on cellular proliferation and apoptosis of CD34+ cells in the ex-vivo culture condition collected from normal bone marrow donors.Methods: We used adenovirus vector containing recombinant antisense telomerase template RNA. Mononuclear cells were obtained from 21 bone marrow donors with normal marrow profile. Each samples were divided into two groups, consisted of the study group treated with telomerase antisense oligonucleotides(TA) and control group without treatment of TA. And then, CD34+ cells were purified from each sample with MACS method and were cultured with LTC-IC method. We evaluated the total and viable cell number, apoptosis degree and blood cell morphology at 2 weeks after LTC-IC culture, and compared between control and experimental groups.Results: Total cell number of study group (2.3–3.3(median:2.6)X104) was not significantly different from that of control group(2.1–3.5(median:2.7)X104). Viable cell percentage of control and study groups were 71–83(median:76)% and 69–84(median:77)%, respectively, without significant difference. Apoptosis degree of study group (25–38%, median:32%) was significantly higher than that of control (13–27%, median 18%), Blood cell morphology of study group was more differentiated without blast cells (0%) than that of control group showing blast cells (4–9%, median 7%) at 2 weeks of culture.Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA might accelerate the differentiation of hematopoietic stem cells with significant effect on the apoptosis degree. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.

Alphaviruses and their Derived Vectors as Anti-Tumor Agents

The alphaviruses Semliki Forest virus (SFV) and Sindbis virus have recently been developed as prototype anti-cancer agents. These are RNA-containing enveloped viruses that code for only 9 proteins of unique sequence. The standard recombinant SFV vector system consists of suicide particles containing recombinant RNA. In addition, alphavirus vectors capable of limited multiplication in the host are also being developed. Several strategies are being adopted to construct prototype SFV vectors for cancer treatment. These include: 1) construction of both prophylactic and therapeutic vaccines to stimulate immunity to tumor-associated antigens, 2) use of apoptosis induction to destroy tumor cells, which includes both the use of the inherent apoptosis-inducing ability of the vector and the action of pro-apoptotic genes cloned into the vector, and 3) expression of cytokines and other immunoregulatory proteins by the vector that enhance anti-tumor immune responses and/or inhibit tumor cell growth. This includes the use of cytokines such as IL-12 that target angiogenesis. Sindbis virus appears to have a natural tropism for tumor cells that may allow targeting both of the wild-type virus and the vector. This approach may also be useful for targeting metastases. For SFV, neurovirulence and/or neurotropism, as well as other tissue damage, may preclude the use of unmodified replication competent wild-type virus in tumor treatment. However, it may be possible to use such a virus in animals that have been vaccinated, using a vector-derived vaccine.

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K(m) for GTP of 3.5 microM, all of the mutations except one had K(m)s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

A novel strategy for creating recombinant infectious RNA virus genomes

Reverse transcriptases with RNase H activity are particularly apt to switch templates and generate recombinant molecules in vitro. This property has been exploited for the first time to create a library of recombinant RNAs 3 between two strains of Cucumber mosaic virus (CMV) or between CMV and Tomato aspermy virus (TAV), which share 75 and 63% sequence identity, respectively. The recombination events were almost entirely of the precise homologous type, and occurred at the same sites as those previously identified in co-infected plants, making it possible to use this strategy to create numerous cDNA fragments with crossovers similar to those occurring in vivo. Sub-cloning of recombinant fragments into an infectious full-length clone was accomplished by homologous recombination in yeast, alleviating the need for in vitro ligation at common restriction sites. Most of the recombinant genomes were infectious. Association of these two methods constitutes an efficient and practical means for generating numerous infectious viral genomes equivalent to ones that might arise by precise homologous recombination between two parental viral genomes in nature.

Coexpression of Gas6/Axl in Human Ovarian Cancers

Objectives: Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, was identified as a ligand for the Axl/Sky family of receptor tyrosine kinases. The aim is to study for the presence of Gas6 and its receptor Axl and Sky related to specific growth in the ovarian cancers, and to evaluate their plausible growth potential and mechanism. Methods: In ovarian cancers of 90 cases, the histoscores and mRNA levels of Gas6, Axl and Sky were determined by immunohistochemistry and competitive reverse transcription-polymerase chain reaction-Southern blot analysis using the recombinant RNA, respectively. Results: The histoscores and mRNA levels of Gas6 and Axl in ovarian cancers were significantly higher than in normal ovaries, regardless of histopathological type or clinical stage of ovarian cancers. Conclusions: Gas6/Axl pathway could play a role in the complex events taking place during the early changes of ovarian cancer progression.

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors.

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.

Clinical Implications of Expression of ETS-1 Related to Angiogenesis in Metastatic Lesions of Ovarian Cancers

Objective: ETS-1 has been identified as a proto-oncogene and a transcription factor for tumor angiogenesis, which is essential for the growth, invasion and metastasis of solid tumors. The aim is to investigate the clinical implications of ETS-1 expression in peritoneal metastatic lesions of ovarian cancers. Methods: In primary tumors and peritoneal metastatic lesions from 30 patients with stage III ovarian cancers, ETS-1 histoscores and ets-1 mRNA levels were determined by immunohistochemistry and competitive RT-PCR-Southern blot analysis using recombinant RNA, respectively. Results: Immunohistochemical staining revealed that ETS-1 was expressed in the cancer cells and vascular endothelial cells. ETS-1 histoscores in the endothelial cells and ets-1 mRNA levels were significantly (p < 0.05) increased in 20 of 30 peritoneal metastatic lesions of ovarian cancers. There was a significant correlation between microvessel counts (MVCs) and ETS-1 histoscores in the endothelial cells (p < 0.001) and between MVCs and ets-1 mRNA levels in the primary tumor and the peritoneal metastatic lesion of ovarian cancers (p < 0.001). Furthermore, the 24-month survival rate of patients with significantly increased ets-1 mRNA level (2/20, 10%) was significantly (p < 0.01) lower than that of patients with no change in the level (6/10, 60%) from the primary tumor to the peritoneal metastatic lesion. Conclusions: ETS-1 might be associated with peritoneal metastasis dominantly as an angiogenic mediator and additionally as an oncogene product to activate tumor invasion in ovarian cancers.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

High-throughput chemiluminometric determination of prostate-specific membrane antigen mRNA in peripheral blood by RT-PCR using a synthetic RNA internal standard.

A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method, employing internal standard (IS) RNA and a simplified chemiluminometric hybridization assay, is described for the determination of prostate-specific membrane antigen (PSMA) mRNA. The recombinant RNA IS has the same binding sites and size as the amplified PSMA mRNA. Biotinylated PCR products (263 bp) from PSMA mRNA and RNA IS are captured in microtiter wells coated with streptavidin, and hybridized with alkaline phosphatase-conjugated probes. The bound alkaline phosphatase (AP) is measured by using a chemiluminogenic substrate. The ratio of the luminescence values obtained for PSMA mRNA and the RNA IS is a linear function of the initial amount of PSMA mRNA present in the sample before RT-PCR. The linear range extends from 500 to 5,000,000 PSMA mRNA copies and the overall reproducibility of the assay, including RT-PCR and hybridization, ranges from 7.4 to 16.6%. Samples containing total RNA from PSMA-expressing LNCaP cells give luminescence ratios linearly related to the number of cells in the range 0.5-5,000 cells.

New non-nucleoside inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

Recombinant RNA-dependent RNA polymerase of hepatitis C virus was purified using a bacterial expression system (Escherichia coli). The system for enzyme activity detection was optimized. The maximum activity was achieved when the reaction was carried out at 30 degrees C in the presence of 3 mM Mg2+ or 0.75 mM Mn2+. Among alpha- and beta-pyrogallaldehydes, effective inhibitors were found. It was shown that they acted at the primer elongation stage, and their binding to the protein is reversible.

Hepatitis C virus infection and non-Hodgkin lymphoma: results of the NCI-SEER multi-center case-control study.

Several studies have noted elevated hepatitis C virus (HCV) prevalence among patients with non-Hodgkin lymphoma (NHL), suggesting that HCV infection increases NHL risk through chronic immune stimulation. Population-based data from the U.S. are lacking. In a population-based case-control study of NHL in the United States, we identified HCV infection using an enzyme immunoassay, confirmed by recombinant immunoblot assay or HCV RNA detection. The association between HCV and NHL was assessed using logistic regression, adjusting for demographic factors, illicit drug use or medical history. Thirty-two of 813 (3.9%) NHL cases and 14 of 684 (2.1%) controls were HCV-infected [odds ratio (OR) 1.96, 95%CI 1.07-4.03]. For separate NHL subtypes, numbers were limited. Nonetheless, positive associations were noted for follicular (OR 2.46, 95%CI 1.01-5.81), marginal zone (3.99, 0-13.6) and mucosa-associated lymphoid tissue (2.04, 0-7.20) NHLs. For all NHLs combined, the HCV-NHL association changed little after adjustment for sex, age, race and study center (OR 1.89, 95%CI 1.00-4.00). HCV was common in controls who had injected drugs (40%) or used other illicit drugs (6.5%), but adjustment for drug use did not affect the HCV-NHL association (OR 1.87, 95%CI 0.95-4.10). Transfusion history was unrelated to HCV status, and adjustment for this exposure did not attenuate the HCV-NHL association (OR 2.15, 95%CI 1.12-4.76). Excluding 4 subjects with a history of hemodialysis or 3 subjects with organ transplants also did not affect the results. Our study demonstrates an association between HCV infection and NHL in the United States. HCV infection may be a cause of NHL.

RNA recombination in vivo in the absence of viral replication.

To study fundamental aspects of RNA recombination, an in vivo RNA recombination system was established. This system allowed the efficient generation of recombinant cytopathogenic pestiviruses after transfection of synthetic, nonreplicatable, subgenomic transcripts in cells infected with a replicating noncytopathogenic virus. Studies addressing the interplay between RNA recombination and replication revealed that cotransfection of noninfected cells with various pairs of nonreplicatable RNA derivatives also led to the emergence of recombinant viral genomes. Remarkably, homologous and nonhomologous recombination occurred between two overlapping transcripts, each lacking different essential parts of the viral RNA-dependent RNA polymerase (RdRp) gene. Apart from the generally accepted viral replicative copy choice recombination, our results prove the existence of a viral RdRp-independent mechanism of RNA recombination that occurs in vivo. It appears likely that such a mechanism not only contributes to the evolution of RNA viruses but also leads to the generation of recombinant cellular RNAs.

Synthesis in Vitro of Rabbit Hemorrhagic Disease Virus Subgenomic RNA by Internal Initiation on (–)Sense Genomic RNA: MAPPING OF A SUBGENOMIC PROMOTER

Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3' one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3'-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (-)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (-)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.

Alphavirus vectors for cancer gene therapy (Review)

Alphaviruses have several characteristics that make them attractive as gene therapy vectors such as transient and high-level expression of a heterologous gene. Alphavirus vectors, Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE) have been developed as gene expression vectors. Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in mammalian cells. The alphavirus RNA replication machinery has been engineered for high level heterologous gene expression. Since an RNA virus vector cannot integrate into chromosomal DNA, concerns about cell transformation are reduced. Alphavirus vectors demonstrate promise for the safe tumor-killing and tumor-specific immune responses. Recombinant alphavirus RNA replicons may facilitate gene therapy of cancer.