Packaging Sequence Research Articles

Sequences at gene segment termini inclusive of untranslated regions and partial open reading frames play a critical role in mammalian orthoreovirus S gene packaging.

Mammalian orthoreovirus (MRV) is a prototypic member of the Spinareoviridae family and has ten double-stranded RNA segments. One copy of each segment must be faithfully packaged into the mature virion, and prior literature suggests that nucleotides (nts) at the terminal ends of each gene likely facilitate their packaging. However, little is known about the precise packaging sequences required or how the packaging process is coordinated. Using a novel approach, we have determined that 200 nts at each terminus, inclusive of untranslated regions (UTR) and parts of the open reading frame (ORF), are sufficient for packaging S gene segments (S1-S4) individually and together into replicating virus. Further, we mapped the minimal sequences required for packaging the S1 gene segment into a replicating virus to 25 5' nts and 50 3' nts. The S1 UTRs, while not sufficient, were necessary for efficient packaging, as mutations of the 5' or 3' UTRs led to a complete loss of virus recovery. Using a second novel assay, we determined that 50 5' nts and 50 3' nts of S1 are sufficient to package a non-viral gene segment into MRV. The 5' and 3' termini of the S1 gene are predicted to form a panhandle structure and specific mutations within the stem of the predicted panhandle region led to a significant decrease in viral recovery. Additionally, mutation of six nts that are conserved across the three major serotypes of MRV that are predicted to form an unpaired loop in the S1 3' UTR, led to a complete loss of viral recovery. Overall, our data provide strong experimental proof that MRV packaging signals lie at the terminal ends of the S gene segments and offer support that the sequence requirements for efficient packaging of the S1 segment include a predicted panhandle structure and specific sequences within an unpaired loop in the 3' UTR.

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30-40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.

Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3'-End of the ORF1b.

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3' end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 μM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3' end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

The VEGAS Platform Is Unsuitable for Mammalian Directed Evolution.

Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution campaigns.

Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveal a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary, we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.

HydrAd: A Helper-Dependent Adenovirus Targeting Multiple Immune Pathways for Cancer Immunotherapy.

Simple SummarySolid tumors are highly immunosuppressive and develop multiple inhibitory mechanisms that must be targeted simultaneously for successful cancer immunotherapy. Adenoviral vectors are promising cancer gene therapy vectors due to their inherent ability to stimulate multiple immune pathways. Adenoviruses are well characterized, and their genomes are easily manipulated, allowing for therapeutic transgene expression. Oncolytic adenoviruses are engineered to replicate specifically in malignant cells, resulting in cancer cell lysis. However, oncolytic adenoviral vectors have limited transgene capacity. Helper-dependent adenoviral vectors have been developed with the capability of expressing multiple transgenes through removal of all viral coding sequences. We have developed a helper-dependent platform for cancer immunotherapy and demonstrate expression of up to four functional transgenes. This platform allows us to target tumors with specific inhibitory pathways using our library of immunomodulatory transgenes in a mix-and-match approach for a synchronized cancer immunotherapy strategy.For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.

Integrating operations and marketing decisions to manage perishability risks with target minimum remaining shelf-life available to consumers

We study an aggregate level planning problem at the bottleneck packaging stage of a Make-and-Pack production system of perishable products and integrate sequencing decisions. The perishability risks are accounted for by producing to order with remaining shelf-life (RSL) dependent demand and age-dependent expected product holding cost till dispatch. Products dispatch time determines the Maximum-RSL delivered. Unlike past studies, we examine a target Minimum-RSL that marketing aims to provide. The product demand decreases from its Maximum-RSL to the Minimum-RSL. The problem is modeled as a two-stage stochastic 0–1 integer non-linear programming problem. The first-stage decisions are the inventory of intermediate products, packaging sequence, capacity allocation, Maximum-RSL, and Minimum-RSL. In the second stage, market demand is realized and has a stochastic error component. The stochastic problem is converted into its deterministic equivalent using the Sample-Average-Approximation method. Representing the demand scenarios using Low Discrepancy Sequences and reducing variance using the Antithetic Variates technique provide stable solutions. Sensitivity analysis demonstrates that the integrated-firm improves freshness criteria (Maximum-RSL and Minimum-RSL) by reducing setup time/demand variability/demand satisfaction requirement (shortage cost) or increasing importance of customer service in product freshness (product holding cost). Maximum-RSL increases with increased packaging rate. High demand variability, shortage cost, product holding cost, and low packaging rate promote integrated sequencing-capacity allocation. An efficient heuristic based on the Generalized Benders’ Decomposition method is proposed to solve large-size problems. The heuristic provides a better solution than the DICOPT solver, solves far fewer non-linear programming problems than the Simple Branch-and-Bound solver, with less computational effort.

A Decentralized Framework for Serverless Edge Computing in the Internet of Things

Serverless computing is becoming widely adopted among cloud providers, thus making increasingly popular the Function-as-a-Service (FaaS) programming model, where the developers realize services by packaging sequences of stateless function calls. The current technologies are very well suited to data centers, but cannot provide equally good performance in decentralized environments, such as edge computing systems, which are expected to be typical for Internet of Things (IoT) applications. In this paper, we fill this gap by proposing a framework for efficient dispatching of stateless tasks to in-network executors so as to minimize the response times while exhibiting short- and long-term fairness, also leveraging information from a virtualized network infrastructure when available. Our solution is shown to be simple enough to be installed on devices with limited computational capabilities, such as IoT gateways, especially when using a hierarchical forwarding extension. We evaluate the proposed platform by means of extensive emulation experiments with a prototype implementation in realistic conditions. The results show that it is able to smoothly adapt to the mobility of clients and to the variations of their service request patterns, while coping promptly with network congestion.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence

The SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the N-protein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or non-coronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the N-terminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV N-protein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.

Examination of specific binding activity of packaging sequence RNAs to the SARS‐CoV nucleocapsid by using cell‐based in vivo assay for RNA‐Protein interaction

Severe acute respiratory syndrome coronavirus (SARS‐CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. The SARS‐CoV nucleocapsid protein serves multiple functions in viral replication, transcription and the formation of viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, little is known about the specific interaction between Nucleocapsid protein and packaging RNA sequence in previously. Herein, we exploited a cell‐based translation inhibition assay to elucidate the protein‐RNA interaction in living cells. Our results demonstrated that the packaging signal is pivotal in the specific interaction of the nucleocapsid and viral genomic RNA for incorporation into virions. We identified that the specific conserved RNA sequence of the nsp12 encoding vRNA of SARS‐CoV, and conserved RNAs of other corona viruses are major determinant sequences for the N‐protein interaction, respectively. In addition, using deletion mapping of the N‐protein, we also found that the specific in vivo RNA binding activity of the Nucleocapsid protein of the SARS‐CoV requires both the N‐terminal and C‐terminal domain. These results provide the first evidence for a specific interaction of the SARS‐CoV Nucleocapsid protein with the its viral packaging RNA sequence in vivo that will be useful as a valuable research tool for further detailed studies and providing new targets for antiviral therapies.Support or Funding InformationThis work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF‐2017R1D1A1B03030315).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions.

The genome of influenza A virus is organized into eight ribonucleoproteins, each composed of a distinct RNA segment bound by the viral polymerase and oligomeric viral nucleoprotein. Packaging sequences unique to each RNA segment together with specific nucleoprotein amino acids are thought to ensure the precise incorporation of these eight ribonucleoproteins into single virus particles, and yet the underlying interaction network remains largely unexplored. We show here that the genome packaging mechanism of an H7N7 subtype influenza A virus widely tolerates the mutation of individual packaging sequences in three different RNA segments. However, combinations of these modified RNA segments cause distinct genome packaging defects, marked by the absence of specific RNA segment subsets from the viral particles. Furthermore, we find that combining a single mutated packaging sequence with sets of specific nucleoprotein amino acid substitutions greatly impairs the viral genome packaging process. Along with previous reports, our data propose that influenza A virus uses a redundant and plastic network of RNA-RNA and potentially RNA-nucleoprotein interactions to coordinately incorporate its segmented genome into virions.IMPORTANCE The genome of influenza A virus is organized into eight viral ribonucleoproteins (vRNPs); this provides evolutionary advantages but complicates genome packaging. Although it has been shown that RNA packaging sequences and specific amino acids in the viral nucleoprotein (NP), both components of each vRNP, ensure selective packaging of one copy of each vRNP per virus particle, the required RNA-RNA and RNA-NP interactions remain largely elusive. We identified that the genome packaging mechanism tolerates the mutation of certain individual RNA packaging sequences, while their combined mutation provokes distinct genome packaging defects. Moreover, we found that seven specific amino acid substitutions in NP impair the function of RNA packaging sequences and that this defect is partially restored by another NP amino acid change. Collectively, our data indicate that packaging of the influenza A virus genome is controlled by a redundant and plastic network of RNA/protein interactions, which may facilitate natural reassortment processes.

Abstract 5090: Efficient KO of PD1 into primary T cells using a new non-integrative lentiviral particle expressing CRISPR/Cas9 system

Abstract Gene editing by the CRISPR system shows great promises for gene therapy. Nevertheless, it must now face a number of challenges especially for the development of safe and efficient delivery tools for in vivo, as well as ex vivo gene editing. Cas9 and sgRNA delivery, mediated either by viral vectors (AAV- or Lentivirus-derived) or transfection protocols (chemical or by electroporation) have been largely and efficiently used but they bring major drawbacks incompatible with clinical applications. Indeed, viral vectors can display uncontrolled chromosomal integrations and transfection protocols are known to induce cell toxicity and/or phenotype modifications of the target cells.. Moreover, the CRISPR technology entails a “hit-and-run” mechanism that only requires a transient expression of the nuclease complex. Therefore, achieving an efficient delivery into hard-to-transfect cells, such as T cells, remains challenging and the need for delivery tools that would allow efficient transfer on most cell types without causing any cell damages is essential for downstream therapeutic applications. Here, we present an innovative tool, named LentiFlash, allowing RNA delivery into target cells without any genomic scar. The RNA encapsidation is mediated via an RNA/protein interaction: the respective properties of the MS2 bacteriophage and the lentiviral vectors have been combined to build a non-integrative packaging system in which the wild type HIV packaging sequence is replaced by the MS2 stem-loop repeats and the MS2 coat protein is inserted into the Nucleocapsid. This new vector breaks with all existing systems, as the resulting lentiparticle is able to deliver non-viral coding or non-coding RNA, at high efficiency, into the cytoplasm of any cell type. Transduction of a large range of cells, from immortalized cells to delicate primary cells, such as T cells and hematopoietic stem cells, with LentiFlash shows an efficient, fast and transient expression of proteins and RNA, with no cell phenotype modification. In particular, LentiFlash particles were successfully used to deliver Cas9, alone or in association with an sgRNA not only targeting a reporter gene into immortalized cells, but outstandingly knocking-out the PD-1 gene into primary human T lymphocytes. This new RNA delivery system is an efficient and safe tool for the delivery of CRISPR editing machinery in most cell types without affecting cell viability and phenotype. The transient, RNA-based mechanism of LentiFlash vector, preventing the risk of integration, associated with its ability to utilize lentiviral production platforms already validated in clinical settings, make it a promising tool for CRISPR therapeutic applications. As a matter of fact, beyond gene editing efficiency, safety of delivery tools is a major concern that should be addressed to move forward with CRISPR clinical development. Citation Format: Pascale Bouillé, Régis Gayon, Lucille Lamouroux, Alexandra Iche, Christine Duthoit, Jean-Christophe Pages. Efficient KO of PD1 into primary T cells using a new non-integrative lentiviral particle expressing CRISPR/Cas9 system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5090. doi:10.1158/1538-7445.AM2017-5090

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

Potential inhibition of HIV-1 encapsidation by oligoribonucleotide-dendrimer nanoparticle complexes.

BackgroundEncapsidation, the process during which the genomic RNA of HIV is packaged into viral particles, is an attractive target for antiviral therapy. This study explores a novel nanotechnology-based strategy to inhibit HIV encapsidation by an RNA decoy mechanism. The design of the 16-mer oligoribonucleotide (RNA) decoy is based on the sequence of stem loop 3 (SL3) of the HIV packaging signal (Ψ). Recognition of the packaging signal is essential to the encapsidation process. It is theorized that the decoy RNA, by mimicking the packaging signal, will disrupt HIV packaging if efficiently delivered into lymphocytes by complexation with a carbosilane dendrimer. The aim of the study is to measure the uptake, toxicity, and antiviral activity of the dendrimer–RNA nanocomplex.Materials and methodsA dendriplex was formed between cationic carbosilane dendrimers and the RNA decoy. Uptake of the fluorescein-labeled RNA into MT4 lymphocytes was determined by flow cytometry and confocal microscopy. The cytoprotective effect (50% effective concentration [EC50]) and the effect on HIV replication were determined in vitro by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and viral load measurements, respectively.ResultsFlow cytometry and confocal imaging demonstrated efficient transfection of lymphocytes. The dendriplex containing the Ψ decoy showed some activity (EC50 =3.20 µM, selectivity index =8.4). However, there was no significant suppression of HIV viral load.ConclusionOligoribonucleotide decoys containing SL3 of the packaging sequence are efficiently delivered into lymphocytes by carbosilane dendrimers where they exhibit a modest cytoprotective effect against HIV infection.

Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage

Previously, we have shown that loss of the histone 3 lysine 27 (H3K27) monomethyltransferases ARABIDOPSIS TRITHORAX-RELATED 5 (ATXR5) and ATXR6 (ATXR6) results in the overreplication of heterochromatin. Here we show that the overreplication results in DNA damage and extensive chromocenter remodeling into unique structures we have named "overreplication-associated centers" (RACs). RACs have a highly ordered structure with an outer layer of condensed heterochromatin, an inner layer enriched in the histone variant H2AX, and a low-density core containing foci of phosphorylated H2AX (a marker of double-strand breaks) and the DNA-repair enzyme RAD51. atxr5,6 mutants are strongly affected by mutations in DNA repair, such as ATM and ATR. Because of its dense packaging and repetitive DNA sequence, heterochromatin is a challenging environment in which to repair DNA damage. Previous work in animals has shown that heterochromatic breaks are translocated out of the heterochromatic domain for repair. Our results show that atxr5,6 mutants use a variation on this strategy for repairing heterochromatic DNA damage. Rather than being moved to adjacent euchromatic regions, as in animals, heterochromatin undergoes large-scale remodeling to create a compartment with low chromatin density.

Structural optimization for remote white light-emitting diodes with quantum dots and phosphor: packaging sequence matters.

White light-emitting diodes (WLEDs) with quantum dots (QDs) and phosphor have attracted tremendous attentions due to their excellent color rendering ability. In the packaging process, QDs layer and phosphor-silicone layer tend to be separated to reduce the reabsorption losses, and to maintain the stability of QDs surface ligands. This study investigated the packaging sequence between QDs and phosphor on the optical and thermal performances of WLEDs. The output optical power and PL spectra were measured and analyzed, and the temperature fields were simulated and validated experimentally by infrared thermal imager. It was found that when driven by 60 mA, the QDs-on-phosphor type WLEDs achieved luminous efficiency (LE) of 110 lm/W, with color rendering index (CRI) of Ra = 92 and R9 = 80, while the phosphor-on-QDs type WLEDs demonstrated lower LE of 68 lm/W, with Ra = 57 and R9 = 24. Moreover, the QDs-on-phosphor type WLEDs generated less heat than that of another, consequently the highest temperature in the QDs-on-phosphor type was lower than another, and the temperature difference can reach 12.3°C. Therefore, in terms of packaging sequence, the QDs-on-phosphor type is an optimal packaging architecture for higher optical efficiency, better color rendering ability and lower device temperature.

Pseudo-typed Semliki Forest virus delivers EGFP into neurons.

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

A conserved influenza A virus nucleoprotein code controls specific viral genome packaging.

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions.