Sequence-dependent Mechanism Research Articles

Mechanisms of RNA export and nuclear retention.

With the identification of huge amount of noncoding RNAs in recent years, the concept of RNA localization has extended from traditional mRNA export to RNA export of mRNA and ncRNA as well as nuclear retention of ncRNA. This review aims to summarize the recent findings from studies on the mechanisms of export of different RNAs and nuclear retention of some lncRNAs in higher eukaryotes, with a focus on splicing-dependent TREX recruitment for the export of spliced mRNA and the sequence-dependent mechanism of mRNA export in the absence of splicing. In addition, evidence to support the involvement of m6 A modification in RNA export with the coordination between the methylase complex and TREX complex as well as sequence-dependent nuclear retention of lncRNA is recapitulated. Finally, a model of sequence-dependent RNA localization is proposed along with the many questions that remain to be answered. This article is categorized under: RNA Export and Localization > RNA Localization RNA Export and Localization > Nuclear Export/Import.

The Expression Dynamics of piRNAs Derived From Male Germline piRNA Clusters and Retrotransposons.

In mammals, germ cells produce a class of small regulatory RNAs called PIWI-interacting RNAs or piRNAs, which are 25–32 nucleotides in length. The profile of testicular piRNAs changes during development. The piRNAs detected in fetal testes at embryonic day 13.5 and later are called fetal piRNAs. The piRNAs detected in testes in a period where germ cells do not yet enter the pachytene stage of meiotic prophase I are called pre-pachytene piRNAs, whereas those in testes at later postnatal days are called pachytene piRNAs. Here, to elucidate the exact expression dynamics of these piRNAs during development, we compared piRNAs present in male germ cells at different stages, which were purified by fluorescence-activated cell sorting, and those in embryonic testes. The analysis identified three distinct groups of piRNA clusters: prospermatogonial, early, and late clusters. piRNA length was largely correlated with the repertoire of PIWI-like proteins in respective germ cells; however, the late piRNA clusters tended to generate longer (PIWIL1-type) piRNAs, whereas the early clusters tended to generate shorter (PIWIL2-type) piRNAs, suggesting a cluster- or sequence-dependent mechanism for loading onto PIWI-like proteins. Retrotransposon-derived piRNAs, particularly evolutionary young retrotransposons, were abundantly produced in prospermatogonia, however, their abundance declined as development proceeded. Thus, in later stages, retrotransposon-derived piRNAs were not enriched with those from evolutionary young elements. The results revealed that, depending on the piRNA clusters from which they are derived, longer PIWIL1-type piRNAs are produced earlier, and shorter PIWIL2-type piRNAs remain in a longer period, than previously thought.

Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks.

Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, 'divergent' Gli-binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.

Bayesian Nonparametric Analysis of Transcriptional Processes

Optical tweezers are routinely used to extract dynamics of nucleic acid associated motors, such as RNA polymerase (RNAP). Their ability to provide data with base-pair spatial resolution is the key. It gives us access to information encoded on the sequence-dependent mechanism underlying molecular motor processivity. Of particular interest is the statistics of RNA Polymerase's pause and dwell dependencies on DNA sequence during both the initiation and elongation stages of transcription. Inspired by results suggesting RNA Pol's DNA composition-specific transcriptional mechanism, we combine the highly temporally resolved data from optical traps with the novel statistical analysis methods of Bayesian nonparametrics (BNPs) to draw base-pair level insight on RNA Pol's activity. On account of BNPs' flexibility in recruiting models of RNA Pol dynamics warranted by the data, and RNA Pol's intrinsically complex dynamics, we rigorously constrain the models allowed by BNPs by proposing new modeling strategies inspired by BNP tools scarcely half a decade old.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody.

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

The 5' Untranslated Region of the Capsid Protein 2 Gene of Mink Enteritis Virus Is Essential for Its Expression.

Mink enteritis virus (MEV), as a parvovirus, is among the smallest of the animal DNA viruses. The limited genome leads to multifunctional sequences and complex gene expression regulation. Here, we show that the expression of viral capsid protein 2 (VP2) of MEV requires its 5' untranslated regions (5' UTR) which promote VP2 gene expression at both transcriptional and translational levels. The expression of VP2 was inhibited in several common eukaryotic expression vectors. Our data showed that the 5' UTR of VP2 enhanced capsid gene transcription but not increased stability or promotes nucleocytoplasmic export of VP2 mRNA. Analysis of the functions of 5' UTR fragments showed that the proximal region (nucleotides [nt] 1 to 270; that is, positions +1 to +270 relative to the transcription initiation site, nt 2048 to 2317 of MEV-L) of 5' UTR of VP2 was necessary for VP2 transcription and also promoted the activity of P38 promoter. Unexpectedly, further analysis showed that deletion of the distal region (nt 271 to 653) of the 5' UTR of VP2 almost completely abolished VP2 translation in the presence of P38, whereas the transcription was still induced significantly. Furthermore, using a luciferase reporter bicistronic system, we identified that the 5' UTR had an internal ribosome entry site-like function which could be enhanced by NS1 via the site at nt 382 to 447. Mutation of the 5' UTR in the MEV full-length clones further showed that the 5' UTR was required for VP2 gene expression. Together, our data reveal an undiscovered function of 5' UTR of MEV VP2 in regulating viral gene expression.IMPORTANCE MEV, a parvovirus, causes acute enteritis in mink. In the present report, we describe an untranslated sequence-dependent mechanism by which MEV regulates capsid gene expression. Our results highlight the roles of untranslated sequences in regulating the transcriptional activity of P38 promoter and translation of capsid genes. These data also reveal the possibility of an unusual translation mechanism in capsid protein expression and the multiple functions of nonstructural protein. A better understanding of the gene expression regulation mechanism of this virus will help in the design of new vaccines and targets for antiviral agents against MEV.

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5'-Terminal AUG.

Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coliptrB instead requires an AUG triplet at the 5' terminus of its mRNA. The 5'-terminal AUG (5'-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5'-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5'-uAUG. Additional sequences within the ptrB 5' untranslated region (5'-UTR) work cumulatively with the 5'-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5'-UTRs from other mRNAs with the ptrB 5'-UTR sequence showed a similar dependence on the 5'-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5'-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5'-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5' termini of E. coli mRNAs and the ability of ptrB 5'-UTR regulation to function independently of gene context, the regulatory effects of 5'-uAUGs on downstream CDSs may be widespread throughout the E. coli genome.IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy.

Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471–503, S604–625, and S1164–1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597–603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597–603 and S604–625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597–603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

A suppressive effect of Sp1 recruitment to the first leader 5′ splice site region on L4-22K-mediated activation of the adenovirus major late promoter

Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5′ splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5′ splice site. Binding of Sp1 to the 5′ splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

Arginylation regulates purine nucleotide biosynthesis by enhancing the activity of phosphoribosyl pyrophosphate synthase.

Protein arginylation is an emerging post-translational modification that targets a number of metabolic enzymes, however the mechanisms and downstream effects of this modification are unknown. Here we show that lack of arginylation renders cells vulnerable to purine nucleotide synthesis inhibitors and affects the related glycine and serine biosynthesis pathways. We show that the purine nucleotide biosynthesis enzyme PRPS2 is selectively arginylated, unlike its close homologue PRPS1, and that arginylation of PRPS2 directly facilitates its biological activity. Moreover, selective arginylation of PRPS2 but not PRPS1 is regulated through a coding sequence-dependent mechanism that combines elements of mRNA secondary structure with lysine residues encoded near the N-terminus of PRPS1. This mechanism promotes arginylation-specific degradation of PRPS1 and selective retention of arginylated PRPS2 in vivo. We therefore demonstrate that arginylation affects both the activity and stability of a major metabolic enzyme.

The Role of DNA Shape in Nucleosome Formation and Positioning

Nucleosomes provide the basic unit of compaction in eukaryotic genomes and the mechanisms that dictate their position along a DNA sequence are of central importance to epigenetics. In this work, we use molecular models of DNA and proteins to explore various aspects of nucleosome positioning. First, we show how the affinity of DNA for histones is encoded in its sequence-dependent shape, including subtle deviations from ideal straight B-DNA and local variations in minor groove width. Using high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. Secondly, we examine the sequence-dependent mechanism by which DNA slides to its optimal position around the histone proteins. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome formation.

Mechanisms of CRLF2 Rearrangement with IGH and P2RY8 in B-Cell Acute Lymphoblastic Leukemia.

Abstract 3243Poster Board III-180 IntroductionPrecursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. While the great majority of these children achieve long-term remission, the disease exacts a high mortality in adults, and children with high-risk features. Fusion oncogenes such as BCR-ABL, MLL-AF4, E2A-PBX and TEL-AML1 are present in 60% of B-ALL cases. Cloning of these rearrangements has provided significant insight into the mechanisms involved in their formation. The gene alterations that affect the remaining 40% are poorly understood. We and others have recently identified CRLF2, a subunit of the thymic stromal lymphopoietin receptor, as a novel oncoprotein in B-ALL. Approximately 15% of adult and high-risk pediatric B-ALL that lack other characteristic gene rearrangements overexpress CRLF2, while leukemias with these rearrangements do not. In cases with CRLF2 overexpression, CRLF2 appears to be the driver of STAT activation, either alone or in combination with gain-of-function mutations in Janus Kinases (JAKs). CRLF2 overexpression results from rearrangements involving the CRLF2 locus, which is located in the pseudoautosomal regions on chromosomes X and Y. These rearrangements can either involve translocation with the immunoglobulin heavy chain (IGH) locus (t(X;14)(p22;q32) or t(Y;14)(p11;q32)) or interstitial deletion (del(X)(p22.33p22.33) or del(Y)(p11.32p11.32)). We sought to define the mechanisms of cleavage and repair that mediate these rearrangements. MethodsFor the translocations, we performed a series of polymerase chain reaction (PCR) assays to amplify junctions between IGHJ segments on chr.14 and the region upstream (i.e., centromeric) of CRLF2 on chr.X/Y. For the deletions, we used single nucleotide polymorphism (SNP) arrays and quantitative PCR to define the extent of deletions and then amplified junctions by PCR. ResultsWe successfully amplified IGHJ/CRLF2 translocation junctions from six B-ALL with CRLF2 overexpression. Junctions involved chr.X/Y sequence between 8-16kb upstream of the CRLF2 translation start site. Five of 6 clustered near putative V(D)J recombinase recognition signal sequences (RSS). Additional evidence for involvement of the V(D)J recombinase was identified in all cases, including the presence of nontemplated nucleotides. In contrast, deletions resulted in juxtaposition of the full length CRLF2 coding sequence to P2RY8. A similar event involving t(X;12) that resulted in SOX5 translocation to P2RY8 has been described in a single case of splenic follicular lymphoma. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family and is highly expressed in lymphocytes. ConclusionCRLF2 rearrangements result in overexpression through juxtaposition to alternate transcriptional control elements. While translocations appear to be mediated by aberrant V(D)J recombination, deletions likely involve an alternate sequence-dependent mechanism that targets downstream of the P2RY8 promoter. DisclosuresNo relevant conflicts of interest to declare.

Tests of the Single-hit DNA Damage Model

The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA ( S) surviving doses ( D) of three damaging agents: γ irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (ln S = kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For γ irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the beta2-adrenergic receptor (beta2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the delta-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.

Identification of a Signal Peptide for Unconventional Secretion

Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. In addition to their nuclear cell-autonomous activities, homeoproteins transfer between cells, thanks to two separate steps of secretion and internalization, which both rely on unconventional mechanisms. Internalization is driven by the third helix of the homeodomain (Penetratin) through a non-vesicular and endocytosis-independent mechanism. In contrast, homeoprotein secretion involves vesicular compartments and requires the presence of a sequence of 11 amino acids (Sec sequence) spanning between the second and third helix of the homeodomain. In this study, we report that the SecPen polypeptide, which combines the two identified domains, Penetratin and Sec, bears all of the necessary information to go in and out of cells. We have analyzed key mechanisms and demonstrated that this peptide can efficiently cross a tight junction epithelium.

Specificity in DNA recognition by a peptide from papillomavirus E2 protein

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N 4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (α1E2), which corresponds to the DNA-recognition helix, α-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with α1E2 peptide, and the α1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the α1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in α1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.

Sequence dependence of C5-propynyl-dU,dC-phosphorothioate oligonucleotide inhibition of the human IGF-I receptor: mRNA, protein, and cell growth.

Human keratinocytes are highly responsive to mitogenic and antiapoptotic signaling by the insulin-like growth factor-I receptor (IGF-IR). IGF-IR hyperstimulation is a feature of hyperplastic skin conditions, making the IGF-IR an appealing target for antisense therapeutic intervention. In this study, we used a C5-propynyl-dU,dC-phosphorothioate oligo-2'-deoxyribonucleotide antisense 15-mer to the human IGF-IR mRNA, along with liposome transfection, to inhibit IGF-IR activity in a human keratinocyte cell line and demonstrated potent inhibition of cell growth despite the presence of serum. To investigate the sequence specificity of these effects and to establish the concentration range over which a purely antisense effect could be demonstrated, we introduced 1, 2, 4, 8, and 15 base mismatches into the oligonucleotide and analyzed changes in inhibitory efficacy. In the 10-30 nM concentration range, the introduction of 1 and 2 mismatches into the middle of the 15-mer only modestly affected inhibitory efficacy, whereas >4 mismatches profoundly reduced mRNA, protein, and growth-inhibitory effects. From these results, we conclude that (1) sequence-specific antisense inhibition of IGF-IR activity in keratinocytes is achievable, (2) potent anti-IGF-IR antisense inhibition can be achieved in vitro at concentrations as low as 10 nM, and (3) a sequence-dependent mechanism is likely to underpin the observed in vivo therapeutic effects (Wraight et al. Nat. Biotechnol. 2000;18:521) of these antisense oligonucleotides (AS-ODN) in cutaneous hyperplastic disorders, such as psoriasis.

Cytoplasmic domain signal sequences that mediate transport of varicella-zoster virus gB from the endoplasmic reticulum to the Golgi.

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.

Beta-Cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenic consensus sequence.

SummaryThe ability of cyclodextrins to enhance the antiviral activity of a phosphodiester oligodeoxynucleotide has been investigated. Aff18-mer oligodeoxynucleotide complementary to the initiation region of the mRNA coding for the spike protein and containing the intergenic consensus sequence of an enteric coronavirus has been tested for antiviral action against virus growth in human adenocarcinoma cells. The phosphodiester oligodeoxynucleotide only showed a limited effect on virus growth rate (from 12 to 34% viral inhibition in cells treated with 7.5 to 25 μM oligodeoxynucleotide, respectively, at a multiplicity of infection of 0.1 infectious particle per cell). In the same conditions, the phosphorothioate analogue exhibited stronger antiviral activity, the inhibition increased from 56 to 90%. The inhibitory effect of this analogue was antisense and sequence-specific. Northern blot analysis showed that the sequence-dependent mechanism of action appears to be the inhibition of mRNA transcription. We conclude that the coronavirus intergenic consensus sequence is a good target for an antisense oligonucleotide antiviral action. The properties of the phosphodiester oligonucleotide was improved after its complexation with cyclodextrins. The most important increase of the antiviral activity (90% inhibition) was obtained with only 7.5 μM oligonucleotide complexed to a cyclodextrin derivative, 6-deoxy-6-S-β-D-galactopyranosyl-6-thio-cyclomaltoheptaose in a molar ratio of 1:100. These studies suggest that the use of cyclodextrin derivatives as carrier for phosphodiester oligonucleotides delivery may be an effective method for increasing the therapeutic potential of these compounds in viral infections.