Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enhancer RNAs”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources
PubMed1996

Anti-interferon activity of adenovirus-2-encoded VAI and VAII RNAs in translation in cultured human cells.

The mode of anti-interferon action of VAI and VAII RNAs of adenovirus type 2 (Ad2) was studied by transfecting interferon-alpha (IFN-alpha)-treated KB cells in culture with a plasmid construct containing the VAI or VAII RNA gene and an SV40 promoter-chloramphenicol acetyltransferase (CAT) gene construct as reporter (pSV2-CAT). The longer the treatment of KB cells with IFN-alpha (2,000 IU/ml) lasted, the higher was the inhibition of CAT expression. A maximum of 76% inhibition was attained without pronounced cytotoxicity during 48 h of treatment. The earlier the VAI RNA gene was transfected, the higher was the enhancement of CAT expression. CAT activity increased from 113 to 157% in normal cells and 200-400% in IFN-alpha treated cells, as compared with the corresponding controls without VAI RNA transfection. The level of CAT mRNA was neither appreciably decreased by IFN-alpha treatment, nor detectably increased by VAI or VAII RNA. The effect of VA RNA thus appeared to be on translation rather than on transcription. The relative constancy of the level of CAT mRNA indicated that IFN-alpha inhibition of CAT expression was not due to the activation of RNase L, but due mainly to translational repression. The level of VAII RNA expressed was only 9-13% of that of VAI RNA. Nevertheless, VAII RNA gene transfection stimulated CAT activity to 112% of the control in non-IFN-alpha-treated cells, and 126-182% in IFN-alpha-inhibited cells. When IFN-alpha treatment was started late after VAI RNA cotransfection, CAT expression increased to 169% which was higher than the expression in cotransfected control cells without IFN-alpha treatment. The enhanced level of CAT activity was in remarkable contrast to the IFN-alpha inhibited level of 25% without VA RNA co-transfection when IFN-alpha was added upon seeding. The enhanced CAT activity in cells treated late with IFN-alpha could be ascribed to higher levels of VA RNAs.

Adenoviruses, Human↗
PubMed1985

Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements.

We obtained eight cDNA clones that define five genes whose expression (appearance of transcripts in the cytoplasm) is enhanced when quiescent mouse fibroblasts are stimulated with serum to divide. Two of these clones (designated 49C8 and 16C8) correspond to RNA species that are present in the cytoplasm of quiescent cells at very low levels. After serum stimulation, the level of 16C8 mRNA rose more rapidly than that of 49C8 RNA, reaching a maximum around 6 to 12 h. The data suggest that 49C8 and 16C8 RNAs are induced as a result of independent stimuli. Either fibroblast growth factor or 12-tetradecanoylphorbol-13-acetate alone could induce 16C8 expression almost as effectively as serum; in contrast, 49C8 was not efficiently induced by epidermal growth factor, fibroblast growth factor, insulin, or 12-tetradecanoylphorbol-13-acetate. Inhibitors of transcription and translation diminished the induction of 16C8, while 49C8 expression was sensitive to actinomycin D but not cycloheximide or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. In vitro transcription experiments with isolated nuclei revealed a peak in transcriptional activity of the 16C8 gene at around 3 h after serum stimulation. Sequence analysis of the 49C8 cDNA clone showed greater than 90% homology of a large portion to a consensus rodent B2 repetitive element.

Animals↗
PubMed2001

Emerging technologies for large-scale screening of human tissues and fluids in the study of severe psychiatric disease.

Neuropsychiatric diseases such as schizophrenia and bipolar disorder are major causes of morbidity throughout the world. Despite extensive searches, no single gene, RNA transcript, or protein has been found which can, on its own, account for these disorders. Recently, the availability of genomic tools such as cDNA microarrays, serial analysis of gene expression (SAGE) and large-scale sequencing of cDNA libraries has allowed researchers to assay biological samples for a large number of RNA transcripts. Similarly, proteomic tools allow for the quantitation of a large number of peptides and proteins. These methods include two-dimensional electrophoresis and surface-enhanced laser desorption/ionization (SELDI). We have initiated experiments which apply these techniques to the comparison of RNAs and proteins expressed in clinical samples obtained from individuals with psychiatric diseases and controls. These methods have the potential to identify pathways that are involved in the pathogenesis of complex psychiatric disorders. The characterization of these pathways may allow for the development of new methods for the diagnosis and treatment of schizophrenia, bipolar disorder, and other human psychiatric diseases.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Approaches for the sequence-specific knockdown of mRNA.

Over the past 25 years there have been thousands of published reports describing applications of antisense nucleic acid derivatives for targeted inhibition of gene function. The major classes of antisense agents currently used by investigators for sequence-specific mRNA knockdowns are antisense oligonucleotides (ODNs), ribozymes, DNAzymes and RNA interference (RNAi). Whatever the method, the problems for effective application are remarkably similar: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. These challenges have been in existence from the first attempts to use antisense research tools, and need to be met before any antisense molecule can become widely accepted as a therapeutic agent.

Animals↗
PubMed2026

Multiplexed perturbation enables scalable pooled screens.

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

Humans↗
PubMedDate not supplied

Development of molecular agents for IGF receptor targeting.

The type 1 insulin-like growth factor receptor (IGF1R) is a promising anticancer treatment target, being frequently overexpressed by tumours, and mediating proliferation, motility and apoptosis protection. Design of specific kinase inhibitors is problematic because of homology between the IGF1R and insulin receptor. This obstacle can be circumvented using sequence-specific molecular agents including antisense, triplex and ribozymes. Recent studies indicate that profound sequence-specific IGF1R gene silencing can be induced by small interfering RNAs that mediate RNA interference in mammalian cells. IGF1R downregulation blocks tumour growth and metastasis, and enhances sensitivity to cytotoxic drugs and irradiation. In murine melanoma cells, radiosensitisation is associated with impaired activation of Atm, which is required for initiation of cell cycle checkpoints and DNA repair pathways after double-strand DNA breaks. Furthermore, tumour cells killed in vivo following IGF1R downregulation can provoke an immune response, protecting against tumour rechallenge. After years of studying the role of the IGF system in tumour biology, novel agents for IGF1R targeting will soon be available for clinical testing. This review summarises the development of molecular agents, and considers factors that will influence clinical activity, including the requirement of established tumours for IGF signalling, and the efficacy and toxicity of IGF1R inhibitors.

Animals↗
PubMed1998

Altered alveolar macrophage function in calorie-restricted rats.

Alveolar macrophage functions associated with clearance of bacteria from the lung were assessed in male Fischer 344 rats maintained on a 25% calorie-restricted diet. Calorie-restricted and ad libitum-fed (control) rats were exposed to concentrations of ozone known to compromise phagocytic function of alveolar macrophages. Ozone suppressed alveolar macrophage phagocytosis of latex beads in vitro in ad libitum-fed rats, but not in calorie-restricted rats. In fact, caloric restriction enhanced phagocytic function in both control and ozone-exposed animals. Ad libitum-fed rats exposed to ozone and challenged with Streptococcus zooepidemicus experienced a prolonged infection and influx of polymorphonuclear leukocytes (PMN), whereas calorie-restricted rats exposed to ozone cleared the bacteria in 24 h without an inflammatory response. Bacterial endotoxin-stimulated in vitro production of nitric oxide and tumor necrosis factor (TNF)-alpha as well as expression of TNF-alpha and interleukin-6 messenger RNAs were all lower in alveolar macrophages isolated from calorie-restricted rats. Together, the data suggest that caloric restriction enhances resistance to gram-positive bacteria, while lowering the production of proinflammatory mediators elicited by endotoxin, a component of gram-negative bacteria. Although increased bacterial resistance is considered beneficial, reduction in the lung's ability to induce inflammatory mediators can have both positive and pathophysiologic consequences.

Animals↗
PubMed2001

Intracellular regeneration of glucocorticoids by 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-1 plays a key role in regulation of the hypothalamic-pituitary-adrenal axis: analysis of 11beta-HSD-1-deficient mice.

11beta-Hydroxysteroid dehydrogenases (11beta-HSDs) catalyze interconversion of active corticosterone and inert 11-dehydrocorticosterone, thus regulating glucocorticoid access to intracellular receptors in vivo. 11beta-HSD type 1 is a reductase, locally regenerating active glucocorticoids. To explore the role of this isozyme in the brain, we examined hypothalamic-pituitary-adrenal axis (HPA) regulation in mice homozygous for a targeted disruption of the 11beta-HSD-1 gene. 11beta-HSD-1-deficient mice showed elevated plasma corticosterone and ACTH levels at the diurnal nadir, with a prolonged corticosterone peak, suggesting abnormal HPA control and enhanced circadian HPA drive. Despite elevated corticosterone levels, several hippocampal and hypothalamic glucocorticoid-sensitive messenger RNAs were normally expressed in 11beta-HSD-1-deficient mice, implying reduced effective glucocorticoid activity within neurons. 11beta-HSD-1-deficient mice showed exaggerated ACTH and corticosterone responses to restraint stress, with a delayed fall after stress, suggesting diminished glucocorticoid feedback. Indeed, 11beta-HSD-1-deficient mice were less sensitive to exogenous cortisol suppression of HPA activation. Thus 11beta-HSD-1 amplifies glucocorticoid feedback on the HPA axis and is an important regulator of neuronal glucocorticoid exposure under both basal and stress conditions in vivo.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1982

[Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on the synthesis and movement of RNA in rat liver (author's transl)].

The effects of malotilate (diisoporpyl 1,3-dithiol-2-ylidenemalonate) on the synthesis and movement of RNA in rat liver were studied in order to elucidate the mechanism of protein synthesis induced by this drug. Incorporation of 3H-cytidine into various RNAs was followed to determine the rates of their synthesis. Oral administration of malotilate to rats enhanced the total RNA synthesis, which reached to maximum level 3 hr after administration. At that time, the increases in the incorporation of radioactivities into the total RNA and the nucleotide pool were closely related to the dosages. Malotilate significantly increased the polyribosomal and t-RNA synthesis as well as nuclear RNA, but not the movement of RNA from the nuclei to the cytosol. It is not likely that malotilate activates specific genes in chromatin because only about 0.1 nmole of malotilate and its metabolites per mg DNA was found in chromatin. From these results, a possible mechanism of protein synthesis induced by malotilate can be proposed as follows: malotilate primarily affects the post-transcription process, promoting the RNA synthesis in the nuclear fraction without any changes of DNA synthesis, and these consequent increases of the polyribosomal RNA and t-RNA synthesis result in the enhancement of protein synthesis.

Animals↗
PubMed2003

Fitness of a turnip crinkle virus satellite RNA correlates with a sequence-nonspecific hairpin and flanking sequences that enhance replication and repress the accumulation of virions.

satC, a satellite RNA associated with Turnip crinkle virus (TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions.

Base Sequence↗
PubMed1989

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment.

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.

Base Sequence↗
PubMed2002

Binding and dissociation of human topoisomerase I with hairpin-loop RNAs: implications for the regulation of HIV-1 replication.

Cellular topoisomerase I has been reported to be present in retroviral particles and to enhance viral cDNA synthesis; however, the mechanisms involved remain unknown. In the present study, it has been demonstrated that human topoisomerase I combines with a stem-loop RNA and that the bound topoisomerase I can be dissociated from RNA substrates in the presence of ATP. In addition, in vitro cleaved synthetic RNA bound by topoisomerase I is subsequently relegated when the topoisomerase I is dissociated by ATP. A mechanism is proposed in which human topoisomerase I is carried into virions and regulates the repair of genomic RNA by its ligation activity.

Adenosine Triphosphate↗
PubMed1996

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

Animals↗
PubMed2006

Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways.

The tetraspanin membrane protein CD151 has been suggested to regulate cancer invasion and metastasis by initiating signaling events. The CD151-mediated signaling pathways involved in this regulation remain to be revealed. In this study, we found that stable transfection of CD151 into MelJuSo human melanoma cells lacking CD151 expression significantly increased cell motility, matrix metalloproteinase-9 (MMP-9) expression, and invasiveness. The enhancement of cell motility and MMP-9 expression by CD151 overexpression was abrogated by inhibitors and small interfering RNAs targeted to focal adhesion kinase (FAK), Src, p38 MAPK, and JNK, suggesting an essential role of these signaling components in CD151 signaling pathways. Also, CD151-induced MMP-9 expression was shown to be mediated by c-Jun binding to AP-1 sites in the MMP-9 gene promoter, indicating AP-1 activation by CD151 signaling pathways. Meanwhile, CD151 was found to be associated with alpha(3)beta(1) and alpha(6)beta(1) integrins in MelJuSo cells, and activation of associated integrins was a prerequisite for CD151-stimulated MMP-9 expression and activation of FAK, Src, p38 MAPK, JNK, and c-Jun. Furthermore, CD151 on one cell was shown to bind to neighboring cells expressing CD151, suggesting that CD151 is a homophilic interacting protein. The homophilic interactions of CD151 increased motility and MMP-9 expression of CD151-transfected MelJuSo cells, along with FAK-, Src-, p38 MAPK-, and JNK-mediated activation of c-Jun in an adhesion-dependent manner. Furthermore, C8161 melanoma cells with endogenous CD151 were also shown to respond to homophilic CD151 interactions for the induction of adhesion-dependent activation of FAK, Src, and c-Jun. These results suggest that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through FAK-Src-MAPKs pathways in human melanoma cells, leading to enhanced cell motility and MMP-9 expression.

Antigens, CD↗
PubMed1989

Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure.

Erythropoietin, a plasma glycoprotein produced primarily by the kidney, is a growth and differentiation factor for erythroid progenitor cells. Production of renal erythropoietin is regulated by modulation of mRNA levels in response to changes in tissue oxygenation. Exposure to cobalt, a nonphysiologic stimulus for erythropoietin production, also acts by inducing mRNA accumulation. To determine whether variations in erythropoietin mRNA levels result from enhanced transcription of the erythropoietin gene, in vitro transcription reactions were performed using isolated rat kidney cell nuclei. Quantitation of specific nuclear RNAs labeled during in vitro transcription revealed active erythropoietin gene transcription in kidney nuclei from anemic-hypoxic and cobalt-treated animals while erythropoietin transcriptional activity was undetectable in normal kidney nuclei. Time course studies showed that stimulation of transcription begins between two and four hours following cobalt treatment and parallels the kinetics of mRNA and plasma erythropoietin accumulation. These results indicate that tissue hypoxia and cobalt exposure specifically enhance erythropoietin gene expression. This increase in erythropoietin production is regulated at least in part at the level of gene transcription.

Animals↗
PubMed2006

Capsid protein gene and the type of host plant differentially modulate cell-to-cell movement of cowpea chlorotic mottle virus.

A study was undertaken to measure the rate of coat protein (CP) independent cell-to-cell movement of cowpea chlorotic mottle bromovirus (CCMV) in three different host plants. A CCMV RNA3 variant in which the CP gene was substituted with enhanced green fluorescent protein (C3/DeltaCP-EGFP) was coinoculated to three different host plants with transcripts of wild type RNAs 1 and 2. Comparative analysis of cell-to-cell movement monitored by the EGFP expression at various days post inoculation revealed that the rate of spread varied with the type of host species inoculated: fastest movement was observed in Nicotiana benthamiana while the rate spread was significantly slower in the natural host cowpea. When CP was expressed as EGFP fusion (C3/CP:EGFP) the rate of spread in N. benthamiana and C. quinoa was slower than that was observed in the absence of CP and remained subliminal in cowpea. Analysis of infection foci by confocal laser scanning microscope revealed that localization of CP:EGFP fusion was distinct in N. benthamiana and C.quinoa and accumulated as fluorescent inclusions at the cell periphery. Additional experiments involving coinoculation of either C3/DeltaCP-EGFP or C3/CP:EGFP with heterologous brome mosaic bromovirus (BMV) genomic RNAs 1 and 2 revealed that, in addition to movement protein and CP, viral replicase also influences cell-to-cell spread. The significance of these results in relation to the mechanism of bromovirus movement is discussed.

Bromovirus↗
PubMed2006

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects.

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.

Gene Expression Profiling↗
PubMed2006

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage.

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

Animals↗