An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ ß = 13.5 h) than soluble ACE2 (t½ ß = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

DNA Origami Calibrators for Counting Fluorophores on Single Particles by Flow Cytometry.

Flow cytometry (FCM) is a high-throughput fluorescence-based technique for multiparameter analysis of individual particles, including cells and nanoparticles. Currently, however, FCM does in many cases not permit proper counting of fluorophore-tagged markers on individual particles, due to a lack of tools for translating FCM output intensities into accurate numbers of fluorophores. This lack hinders derivation of detailed biologic information and comparison of data between experiments with FCM. To address this technological void, the authors here use DNA nanotechnology to design and construct barrel-shaped DNA-origami nanobeads for fluorescence/antigen quantification in FCM. Each bead contains a specific number of calibrator fluorophores and a fluorescent trigger domain with an alternative fluorophore for proper detection in FCM. Using electron microscopy, single-particle fluorescence microscopy, and FCM, the design of each particle is verified. To validate that the DNA bead-based FCM calibration enabled the authors to determine the number of antigens on a biological particle, the uniform and well-characterized murine leukemia virus (MLV) is studied. 48 ± 11 envelope surface protein (Env) trimers per MLV is obtained, which is consistent with reported numbers that relied on low-throughput imaging. Thus, the authors' DNA-beads should accelerate quantitative studies of the biology of individual particles with FCM.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

A new DNA sensor system for specific and quantitative detection of mycobacteria.

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.

The Effects of Dithiothreitol on DNA.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

Novel DNA sensor system for highly sensitive and quantitative retrovirus detection using virus encoded integrase as a biomarker.

In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions happening within nanometer dimensions to directly detectable micrometer sized DNA products. The system utilizes the unique integration reaction of IN to generate a surface anchored nicked DNA circle that serves as a substrate for rolling circle amplification and allows for specific, quantitative and sensitive detection of purified recombinant IN or virus particles with a detection limit of less than 30 virus particles per µL of sample. Moreover, by modifying the nucleotide sequences of the utilized DNA it was possible to tailor the system to distinguish between the highly pathogenic lentivirus HIV and the gammaretrovirus murine leukemia virus present in a given sample. Infections with HIV remain a major threat to global health with more than 2 million new infections and 1 million deaths each year. The sensitive and specific detection of HIV particles based on IN activity holds promise for the development of a new type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment.

Utility of next-generation RNA-sequencing in identifying chimeric transcription involving human endogenous retroviruses.

Several studies have shown that human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively HERVs) impose direct regulation on human genes through enhancer and promoter motifs present in their long terminal repeats (LTRs). Although chimeric transcription in which novel gene isoforms containing retroviral and human sequence are transcribed from viral promoters are commonly associated with disease, regulation by HERVs is beneficial in other settings; for example, in human testis chimeric isoforms of TP63 induced by an ERV9 LTR protect the male germ line upon DNA damage by inducing apoptosis, whereas in the human globin locus the γ- and ß-globin switch during normal hematopoiesis is mediated by complex interactions of an ERV9 LTR and surrounding human sequence. The advent of deep sequencing or next-generation sequencing (NGS) has revolutionized the way researchers solve important scientific questions and develop novel hypotheses in relation to human genome regulation. We recently applied next-generation paired-end RNA-sequencing (RNA-seq) together with chromatin immunoprecipitation with sequencing (ChIP-seq) to examine ERV9 chimeric transcription in human reference cell lines from Encyclopedia of DNA Elements (ENCODE). This led to the discovery of advanced regulation mechanisms by ERV9s and other HERVs across numerous human loci including transcription of large gene-unannotated genomic regions, as well as cooperative regulation by multiple HERVs and non-LTR repeats such as Alu elements. In this article, well-established examples of human gene regulation by HERVs are reviewed followed by a description of paired-end RNA-seq, and its application in identifying chimeric transcription genome-widely. Based on integrative analyses of RNA-seq and ChIP-seq, data we then present novel examples of regulation by ERV9s of tumor suppressor genes CADM2 and SEMA3A, as well as transcription of an unannotated region. Taken together, this article highlights the high suitability of contemporary sequencing methods in future analyses of human biology in relation to evolutionary acquired retroviruses in the human genome.

Immune suppressive activity of the influenza fusion peptide.

Immune suppressive domains have been identified in retro and filoviral fusion proteins. Such domains constitute small peptide motifs that are evolutionarily very well preserved within each group. We here test the hypothesis that such preservation reflects a dual selection pressure for both immune suppression and membrane fusion activity in influenza viruses for which no immune suppressive peptide motifs have been identified. We identified a conserved motif in the fusion peptide of influenza hemagglutinin as a candidate for an immune suppressive domain using comparative and phylogenetic analysis. This peptide was indeed found to exhibit immune suppressive activity in several in vitro assays. Similar to the previously reported peptides from retro and filoviruses the influenza peptide had immune suppressive activity when presented as a dimer but not as a monomer.

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities.

UNLABELLED: We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE: Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry.

Two Virus-Induced MicroRNAs Known Only from Teleost Fishes Are Orthologues of MicroRNAs Involved in Cell Cycle Control in Humans.

MicroRNAs (miRNAs) are ~22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene regulation mediated by miRNAs has been shown to be involved in almost all biological phenomena, including development, metabolism, cell cycle, tumor formation, and host-pathogen interactions. To address the latter in a primitive vertebrate host, we here used an array platform to analyze the miRNA response in rainbow trout (Oncorhynchus mykiss) following inoculation with the virulent fish rhabdovirus Viral hemorrhagic septicaemia virus. Two clustered miRNAs, miR-462 and miR-731 (herein referred to as miR-462 cluster), described only in teleost fishes, were found to be strongly upregulated, indicating their involvement in fish-virus interactions. We searched for homologues of the two teleost miRNAs in other vertebrate species and investigated whether findings related to ours have been reported for these homologues. Gene synteny analysis along with gene sequence conservation suggested that the teleost fish miR-462 and miR-731 had evolved from the ancestral miR-191 and miR-425 (herein called miR-191 cluster), respectively. Whereas the miR-462 cluster locus is found between two protein-coding genes (intergenic) in teleost fish genomes, the miR-191 cluster locus is found within an intron of a protein-coding gene (intragenic) in the human genome. Interferon (IFN)-inducible and immune-related promoter elements found upstream of the teleost miR-462 cluster locus suggested roles in immune responses to viral pathogens in fish, while in humans, the miR-191 cluster functionally associated with cell cycle regulation. Stimulation of fish cell cultures with the IFN inducer poly I:C accordingly upregulated the expression of miR-462 and miR-731, while no stimulatory effect on miR-191 and miR-425 expression was observed in human cell lines. Despite high sequence conservation, evolution has thus resulted in different regulation and presumably also different functional roles of these orthologous miRNA clusters in different vertebrate lineages.

Human endogenous retroviruses sustain complex and cooperative regulation of gene-containing loci and unannotated megabase-sized regions.

BACKGROUND: Evidence suggests that some human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively ERVs) regulate the expression of neighboring genes in normal and disease states; e.g. the human globin locus is regulated by an ERV9 that coordinates long-range gene switching during hematopoiesis and activates also intergenic transcripts. While complex transcription regulation is associated with integration of certain exogenous retroviruses, comparable regulation sustained by ERVs is less understood. FINDINGS: We analyzed ERV transcription using ERV9 consensus sequences and publically available RNA-sequencing, chromatin immunoprecipitation with sequencing (ChIP-seq) and cap analysis gene expression (CAGE) data from ENCODE. We discovered previously undescribed and advanced transcription regulation mechanisms in several human reference cell lines. We show that regulation by ERVs involves long-ranging activations including complex RNA splicing patterns, and transcription of large unannotated regions ranging in size from several hundred kb to around 1 Mb. Moreover, regulation was found to be cooperatively sustained in some loci by multiple ERVs and also non-LTR repeats. CONCLUSION: Our analyses show that endogenous retroviruses sustain advanced transcription regulation in human cell lines, which shows similarities to complex insertional mutagenesis effects exerted by exogenous retroviruses. By exposing previously undescribed regulation effects, this study should prove useful for understanding fundamental transcription mechanisms resulting from evolutionary acquisition of retroviral sequence in the human genome.

Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors.

BACKGROUND: Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. RESULTS: We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. CONCLUSIONS: We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.

Gene expression profiling of murine T-cell lymphoblastic lymphoma identifies deregulation of S-phase initiating genes.

In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs.

Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization.

Many cytokine receptors are cell surface proteins that promiscuously combine to form active signalling homo- or heterodimers. Thus, receptor chain dimerization can be viewed as a direct measure of a high probability of intracellular signalling by specific cytokines. Proximity ligation assay (PLA) is an antibody-based method for selective and highly sensitive detection of protein interactions by microscopy. As proof of concept, the aim of this study was to combine antibodies towards interleukin 7 receptor alpha (IL-7Rα) and the common gamma chain (γc) with PLA and flow cytometry to enable the detection of IL-7 receptor heterodimers. The presence of IL-7 receptor heterodimers on the surface of the HPB-ALL T cell line was detected by PLA and microscopy with a resolution of one complex per cell. Optimisation of the PLA reaction on cell suspensions identified buffer effects with critical importance for the flow cytometric outcome. In addition, blocking, fixation and incubation conditions were optimised to prevent unspecific antibody binding. PLA combined with flow cytometry very sensitively detected receptor heterodimers on the cell surface. Thus, the method is a powerful tool for the investigation of cytokine receptor dimerization.

Septin9 is involved in T-cell development and CD8+ T-cell homeostasis.

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.

Deregulated Nras expression in knock-in animals harboring a gammaretroviral long terminal repeat at the Nras/Csde1 locus.

To investigate mechanisms and phenotypic effects of insertional mutagenesis by gammaretroviruses, we have developed mouse lines containing a single Akv 1-99 long terminal repeat (LTR) and a floxed PGK/Tn5 neomycin cassette at the Nras proto-oncogene at positions previously identified as viral integration sites in Akv 1-99 induced tumors. The insert did not compromise the embryonic development, however, the cassette had an effect on Nras expression in all tissues analyzed. Cre-mediated excision of the PGK/Tn5 neomycin cassette in two of the lines caused upregulation of Nras. Altogether, the knock-in alleles are characterized by modulation of expression of the target gene from more than ten-fold upregulation to three-fold downregulation and exemplify various mechanisms of deregulation by insertional mutagenesis. LTR knock-in mice may serve as a tool to investigate mechanisms of retroviral insertional mutagenesis and as a way of constitutive or induced modulation of expression of a target gene.

Epigenetic marking and repression of porcine endogenous retroviruses.

Endogenous retroviruses (ERVs) are remnants of retroviral germ line infections and have been identified in all mammals investigated so far. Although the majority of ERVs are degenerated, some mammalian species, such as mice and pigs, carry replication-competent ERVs capable of forming infectious viral particles. In mice, ERVs are silenced by DNA methylation and histone modifications and some exogenous retroviruses were shown to be transcriptionally repressed after integration by a primer-binding site (PBS) targeting mechanism. However, epigenetic repression of porcine ERVs (PERVs) has remained largely unexplored so far. In this study, we screened the pig genome for PERVs using LTRharvest, a tool for de novo detection of ERVs, and investigated various aspects of epigenetic repression of three unrelated PERV families. We found that these PERV families are differentially up- or downregulated upon chemical inhibition of DNA methylation and histone deacetylation in cultured porcine cells. Furthermore, chromatin immunoprecipitation analysis revealed repressive histone methylation marks at PERV loci in primary porcine embryonic germ cells and immortalized embryonic kidney cells. PERV elements belonging to the PERV-γ1 family, which is the only known PERV family that has remained active up to the present, were marked by significantly higher levels of histone methylations than PERV-γ2 and PERV-ß3 proviruses. Finally, we tested three PERV-associated PBS sequences for repression activity in murine and porcine cells using retroviral transduction experiments and showed that none of these PBS sequences induced immediate transcriptional silencing in the tested primary porcine cells.

Nras overexpression results in granulocytosis, T-cell expansion and early lethality in mice.

NRAS is a proto-oncogene involved in numerous myeloid malignancies. Here, we report on a mouse line bearing a single retroviral long terminal repeat inserted into Nras. This genetic modification resulted in an increased level of wild type Nras mRNA giving the possibility of studying the function and activation of wild type NRAS. Flow cytometry was used to show a variable but significant increase of immature myeloid cells in spleen and thymus, and of T-cells in the spleen. At an age of one week, homozygous mice began to retard compared to their wild type and heterozygous littermates. Two weeks after birth, animals started to progressively lose weight and die before weaning. Heterozygous mice showed a moderate increase of T-cells and granulocytes but survived to adulthood and were fertile. In homozygous and heterozygous mice Gfi1 and Gcsf mRNA levels were upregulated, possibly explaining the increment in immature myeloid cells detected in these mice. The short latency period indicates that Nras overexpression alone is sufficient to cause dose-dependent granulocytosis and T-cell expansion.

Functional selection of shRNA loops from randomized retroviral libraries.

Gene silencing by RNA interference (RNAi) can be achieved by the ectopic expression of tailored short hairpin RNAs (shRNAs) which after export to the cytoplasm are processed by Dicer and incorporated into the RNA induced silencing complex (RISC). Design rules for shRNAs have been the focus of several studies, but only a few reports have turned the attention to the sequence of the loop-region. In this work we selected high-functional and low-functional shRNA loops from retroviral hairpin-loop-libraries in an RNAi reporter assay. The procedure revealed a very significant and stem sequence-dependent effect of the loop on shRNA function and although neither strong consensus loop sequence nor structural motifs could be identified, a preferred loop sequence (5'-UGUGCUU-3') was found to support robust knock down with little stem sequence dependency. These findings will serve as a guide for designing shRNAs with improved knock down capacity.

Construction of a gammaretrovirus with a novel tropism and wild-type replication kinetics capable of using human APJ as entry receptor.

We have constructed a replication-competent gammaretrovirus (SL3-AP) capable of using the human G-protein-coupled receptor hAPJ as its entry receptor. The envelope protein of the virus was made by insertion of the 13-amino-acid peptide ligand for hAPJ, flanked by linker sequences, into one of the variable loops of the receptor binding domain of SL3-2, a murine leukemia virus (MLV) that uses the xenotropic-polytropic virus receptor Xpr1 and which has a host range limited to murine cells. This envelope protein can utilize hAPJ as well as murine Xpr1 for entry into host cells with equal efficiencies. In addition, the SL3-AP virus replicates in cells expressing either of its receptors, hAPJ and murine Xpr1, and causes resistance to superinfection and downregulation of hAPJ in infected cells. Thus, SL3-AP is the first example of a retargeted replication-competent retrovirus, with replication characteristics and receptor interference properties similar to those of natural isolates.