The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial ...attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.
As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely ...to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection.
Human cytomegalovirus infection is a significant cause of disease in immunocompromised populations, individuals with heart disease, and recipients of solid organ and bone marrow transplants. HCMV is also the leading cause of infectious congenital birth defects. The majority of antivirals in clinical use target components of the virus to specifically inhibit replication. However, a major drawback of this approach is the emergence of resistance. An alternative approach is to target host factors that the virus requires for successful infection. In this study, multiple host factors were identified that were found to be essential for the production of newly infectious human cytomegalovirus. Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate and how cells function and provides potential targets for novel antivirals.
In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they ...migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelialto-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3β) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3β, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3βresistant Smad1 resulted in aberrant CPC migration trajectories. Derepression of GSK3β by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.
Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a ...dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.
Composer, pianist and photographer, Martial Caillebotte (1853–1910) was the unknown younger brother of famous Impressionist painter, Gustave Caillebotte. Martial studied piano and harmony at the ...Paris Conservatory from 1870–1874. Martial created a substantial number of musical compositions including mélodies, scènes lyriques, operas, symphonic poems, as well as sacred choral and symphonic works. Born into a wealthy Parisian family, he did not need to work for a living and did not self-promote, therefore his pieces were rarely performed and after his death, most of his compositions were left in family archives. In the late 1990’s a rebirth of interest in the Caillebotte name (both Gustave and Martial) and artistic contributions led to the rediscovery of Martial Caillebotte’s music and current research.There is little scholarly work written on Martial Caillebotte, therefore this dissertation is meant to serve as an in-depth academic study and presentation of Martial Caillebotte and his works. Chapter 1 provides a biographical and historical examination of Caillebotte’s life and musical influences. A listing and description of his compositions are provided along with corresponding reviews and historical journal announcements. Chapter 2 is dedicated to the poets that Caillebotte set to music: Théophile Gautier (1811–1872), Pierre de Ronsard (1524–1585), Olivier Basselin (1400–1450), Félix Arvers (1806–1850), L. Gilis (dates unknown), and Alfred de Musset (1810–1857). Chapter 3 presents analytical essays and performance practice suggestions for Caillebotte’s mélodies and scènes lyriques for voice and piano: “Sérénade,” Mignonne, allons voir si la rose,” “Chanson,” “Sonnet,” “Le Nuage,” and “Attente.” The analytical essays are presented in the following outline: Text of the poem in French/translations in English; poem analysis; Caillebotte’s setting of the poetry; and musical analysis. A full score as well as musical examples are provided for each mélodie. Two appendices provide information on the three recordings by Chœur Régional Vittoria Île-de-France of Martial Caillebotte’s choral works (2012, 2014, and 2017) and chronological listings of the 21st century live performances of Martial Caillebotte’s compositions.
•Partial lipodystrophy is an unusual presentation of Werner syndrome.•Exome analysis revealed a novel splice site variant c.2732+5G>A.•c.2732+5G>A leads to exon skipping and partially abrogates the ...WRN helicase domain.•The WRN gene should be included in different gene panels to prompt diagnosis.
Werner syndrome is a premature ageing disorder caused by biallelic variants in the WRN gene. WRN encodes a dual DNA helicase/exonuclease enzyme. Molecular diagnosis is commonly only made at a late disease stage in the third or fourth decade, when cardinal features have become apparent. We describe a 28 year-old woman who presented with early onset diabetes associated with partial lipodystrophy, severe dyslipidaemia and rapidly progressive liver fibrosis related to non-alcoholic steatohepatitis in the absence of progeroid features. Werner syndrome was diagnosed by trio exome analysis, which revealed compound heterozygous WRN mutations: the known variant c.1290_1293del (p.Asn430Lysfs*7) and the novel intronic splice site variant c.2732+5G>A. cDNA analysis demonstrated this to lead to in-frame skipping of exon 22, predicted to delete most of the zinc binding region of the helicase domain. We suggest that including the WRN gene in genetic analysis of early onset diabetes, lipodystrophy or dyslipidaemia would allow for the opportunity to diagnose some cases of Werner syndrome long before clinical criteria are met, thereby allowing early implementation of important primary prevention interventions.
As a first line of defence, the interferon-mediated innate immune response is pivotal to protect cells against invading pathogens. At the heart of it are hundreds of interferon-stimulated genes ...(ISGs) upregulated substantially shortly after viral infection. Some of these ISGs act indirectly to interfere with virus life cycle via regulation of interferon signalling pathways whereas other ISG can act directly to inhibit viral replication via interaction with viral components. Although many of the ISGs have been identified decades ago, only a handful of them have been characterized about their antiviral activity. Here, we used an arrayed expression lentivirus library of more than 400 ISGs to identify the ISGs with anti-HCMV activity. We performed parallel screens using wild type fibroblast cells and IRF3 KO fibroblast cells generated by CRISPR/Cas9 editing to identify ISGs more likely to directly inhibit HCMV, as opposed to activation of IFN signalling. The IRF3-independent ISGs identified in the screen include those that signal through IRF3 independent pathways such as IRF7 or known to inhibit HCMV directly such as IDO, RIPK2 and AIM2 validating the screening approach. Interestingly, we also identified novel IRF3-independent anti-HCMV ISGs, indicating they may play a role in directly inhibiting the virus.
Abstract Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral ...gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases.
Abstract Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory ...responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.
The heart, the first organ to function in developing vertebrate embryos1, is affected by congenital defects in roughly 1% of the worldwide human population. These abnormalities frequently require ...treatment for the entire lifespan of the affected individual2,3. microRNAs are an exciting class of non-coding regulatory genes that are known to be essential for the heart development of many organisms4,5. A miRNA becomes incorporated into the miRNA-induced silencing complex, which binds to a target mRNA transcript and inhibits its translation. Dysregulation of miRNAs is associated with failing human hearts6. More than 2,500 mature human miRNAs are currently annotated, and with each miRNA predicted to potentially target several hundred transcripts, the further study of these molecules is vital for the complete understanding of cardiac biology. A miRNA expression profile in the chick heart, a model of human heart development, over several stages of embryonic development was generated by next generation sequencing. Two novel chick miRNAs were discovered, and 31 known animal miRNAs not yet annotated in the chick genome were discerned. The predicted targets of three miRNAs thought to be essential for heart development, miR-130, miR-138, and miR-499, were examined in vitro. Twelve genes were confirmed as targets of at least one of these three miRNAs by luciferase reporter assay, including genes in the Wnt signalling pathway which is essential at specific stages of heart development7. The roles of miR-1, known to be indispensable for heart formation and function, were also studied in the developing chick heart. Two targets of miR-1, FZD7 and RARβ, had been identified in vitro8. This study showed that inhibition of miR-1 in the HH14 chick resulted increased FZD7 and RARβ protein levels, as well as in delayed development, possibly due to a loss of inhibition of canonical Wnt signalling in the epicardium.
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