Publications by authors named "Edward M Kennedy"

53 Publications

Epitranscriptomic addition of mA regulates HIV-1 RNA stability and alternative splicing.

Genes Dev 2021 Jul 17;35(13-14):992-1004. Epub 2021 Jun 17.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

Previous work has demonstrated that the epitranscriptomic addition of mA to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that mA function is largely mediated by cellular mA binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that mA addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear mA reader YTHDC1 and the cytoplasmic mA reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to mA residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to mA sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.348508.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247604PMC
July 2021

ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

Cancer Immunol Res 2021 03 22;9(3):291-308. Epub 2020 Dec 22.

Oncorus, Inc., Cambridge, Massachusetts.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8 T-cell responses. , biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-20-0609DOI Listing
March 2021

Design of an Interferon-Resistant Oncolytic HSV-1 Incorporating Redundant Safety Modalities for Improved Tolerability.

Mol Ther Oncolytics 2020 Sep 8;18:476-490. Epub 2020 Aug 8.

Oncorus, Inc., Cambridge, MA, USA.

Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. , this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omto.2020.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479328PMC
September 2020

Epitranscriptomic Addition of mC to HIV-1 Transcripts Regulates Viral Gene Expression.

Cell Host Microbe 2019 08;26(2):217-227.e6

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (mC) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for mC on HIV-1 RNAs. NSUN2 inactivation inhibits not only mC addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of mC dysregulates the alternative splicing of viral RNAs. These data identify mC as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2019.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714563PMC
August 2019

Chemical, Biological, Radiological, Nuclear, and Explosive (CBRNE) Science and the CBRNE Science Medical Operations Science Support Expert (CMOSSE).

Disaster Med Public Health Prep 2019 12;13(5-6):995-1010

National Center for Disaster Preparedness, Earth Institute at Columbia University, New York, New York.

A national need is to prepare for and respond to accidental or intentional disasters categorized as chemical, biological, radiological, nuclear, or explosive (CBRNE). These incidents require specific subject-matter expertise, yet have commonalities. We identify 7 core elements comprising CBRNE science that require integration for effective preparedness planning and public health and medical response and recovery. These core elements are (1) basic and clinical sciences, (2) modeling and systems management, (3) planning, (4) response and incident management, (5) recovery and resilience, (6) lessons learned, and (7) continuous improvement. A key feature is the ability of relevant subject matter experts to integrate information into response operations. We propose the CBRNE medical operations science support expert as a professional who (1) understands that CBRNE incidents require an integrated systems approach, (2) understands the key functions and contributions of CBRNE science practitioners, (3) helps direct strategic and tactical CBRNE planning and responses through first-hand experience, and (4) provides advice to senior decision-makers managing response activities. Recognition of both CBRNE science as a distinct competency and the establishment of the CBRNE medical operations science support expert informs the public of the enormous progress made, broadcasts opportunities for new talent, and enhances the sophistication and analytic expertise of senior managers planning for and responding to CBRNE incidents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/dmp.2018.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334863PMC
December 2019

Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.

mBio 2019 06 11;10(3). Epub 2019 Jun 11.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of -methyladenosine (mA), 5-methylcytosine (mC), and 2'O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of mA and mC residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for mA and mC in viral replication, we also demonstrate that overexpression of the key mA reader protein YTHDF2 enhances MLV replication, while downregulation of the mC writer NSUN2 inhibits MLV replication. The data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications mA, mC, and Nm, suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these mA or mC modifications from MLV transcripts inhibits MLV replication in , and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.01209-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561033PMC
June 2019

Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth .

Future Virol 2018 Jul 12;13(7):475-482. Epub 2018 Jun 12.

Departments of Medicine & Molecular Genetics & Microbiology, Duke Cancer Institute, Duke University Medical Center, Durham, NC 27710, USA.

Aim: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor .

Materials & Methods: We grew HPV16, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs.

Results & Conclusion: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/fvl-2018-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136077PMC
July 2018

Influenza A virus-derived siRNAs increase in the absence of NS1 yet fail to inhibit virus replication.

RNA 2018 09 14;24(9):1172-1182. Epub 2018 Jun 14.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaute 2, fails to enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells. Indeed, a number of mammalian VSRs have been described, of which the most prominent is the influenza A virus (IAV) NS1 protein, which has not only been reported to inhibit RNAi in plants and insects but also to prevent the production of viral siRNAs in IAV-infected human cells. Here, we confirm that an IAV mutant lacking NS1 indeed differs from wild-type IAV in that it induces the production of readily detectable levels of Dicer-dependent viral siRNAs in infected human cells. However, we also demonstrate that these siRNAs have little if any inhibitory effect on IAV gene expression. This is likely due, at least in part, to their inefficient loading into RNA-induced silencing complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.066332.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097656PMC
September 2018

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

Cell Host Microbe 2017 Sep;22(3):377-386.e5

Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

Many viral RNAs are modified by methylation of the N position of adenosine (mA). mA is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses mA-modified RNAs, but the effects of mA on this segmented RNA virus remain unclear. We demonstrate that global inhibition of mA addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular mA "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether mA residues modulate IAV RNA function in cis, we mapped mA residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate mA on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, mA residues in IAV transcripts enhance viral gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2017.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615858PMC
September 2017

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor.

Cell Rep 2017 08;20(7):1503-1512

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA. Electronic address:

Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.07.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568676PMC
August 2017

A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs.

RNA Biol 2017 11 21;14(11):1570-1579. Epub 2017 Jul 21.

a Department of Molecular Genetics & Microbiology and Center for Virology , Duke University Medical Center , Durham , North Carolina , USA.

While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15476286.2017.1334755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785219PMC
November 2017

Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

Cell 2017 03;169(1):72-84.e13

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA; Center for RNA Biology, Duke University, Durham, NC 27710, USA; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address:

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5456452PMC
March 2017

Viral Epitranscriptomics.

J Virol 2017 05 13;91(9). Epub 2017 Apr 13.

Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina, USA

Although it has been known for over 40 years that eukaryotic mRNAs bear internal base modifications, it is only in the last 5 years that the importance of these modifications has begun to come into focus. The most common mRNA modification, the addition of a methyl group to the position of adenosine (mA), has been shown to affect splicing, translation, and stability, and mA is also essential for embryonic development in organisms ranging from plants to mice. While all viral transcripts examined so far have been found to be extensively mA modified, the role, if any, of mA in regulating viral gene expression and replication was previously unknown. However, recent data generated using HIV-1 as a model system strongly suggest that sites of mA addition not only are evolutionarily conserved but also enhance virus replication. It is therefore likely that the field of viral epitranscriptomics, which can be defined as the study of functionally relevant posttranscriptional modifications of viral RNA transcripts that do not change the nucleotide sequence of that RNA, is poised for a major expansion in scientific interest and may well fundamentally change our understanding of how viral replication is regulated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02263-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391447PMC
May 2017

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity.

mBio 2017 01 17;8(1). Epub 2017 Jan 17.

Center for Virology and Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA

Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4 T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.

Importance: The genomes of RNA viruses have the potential to interact with cellular miRNAs, which could lead to their incorporation into virions, with unknown effects on virion function. Here, it is demonstrated that wild-type HIV-1 virions essentially randomly incorporate low levels of the miRNAs expressed by infected cells. However, the specific incorporation of high levels of individual cellular miRNAs can be induced by insertion of cognate target sites into the viral genome. Of note, this results in a modest but significant inhibition of virion infectivity. These data imply that cellular miRNAs have the potential to inhibit viral replication by interfering with not only viral mRNA function but also virion infectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.02125-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241401PMC
January 2017

Partial reconstitution of the RNAi response in human cells using Drosophila gene products.

RNA 2017 02 11;23(2):153-160. Epub 2016 Nov 11.

Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.059345.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238790PMC
February 2017

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

Cell Host Microbe 2016 Nov 20;20(5):654-665. Epub 2016 Oct 20.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA; Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

The RNA modification N6-methyladenosine (mA) post-transcriptionally regulates RNA function. The cellular machinery that controls mA includes methyltransferases and demethylases that add or remove this modification, as well as mA-binding YTHDF proteins that promote the translation or degradation of mA-modified mRNA. We demonstrate that mA modulates infection by hepatitis C virus (HCV). Depletion of mA methyltransferases or an mA demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped mA sites across the HCV genome and determined that inactivating mA in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of mA on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by mA. Altogether, this work identifies mA as a conserved regulatory mark across Flaviviridae genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2016.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123813PMC
November 2016

Gene Editing: A New Tool for Viral Disease.

Annu Rev Med 2017 01 26;68:401-411. Epub 2016 Aug 26.

Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710; email: ,

The emergence of the CRISPR/Cas system of antiviral adaptive immunity in bacteria as a facile system for gene editing in mammalian cells may well lead to gene editing becoming a novel treatment for a range of human diseases, especially those caused by deleterious germline mutations. Another potential target for gene editing are DNA viruses that cause chronic pathogenic diseases that cannot be cured by using currently available drugs. We review the current state of this field and discuss the potential advantages and problems with using a gene editing approach as a treatment for diseases caused by DNA viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-med-051215-031129DOI Listing
January 2017

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells.

Virology 2016 08 14;495:92-100. Epub 2016 May 14.

Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA. Electronic address:

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G1/G0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2016.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912869PMC
August 2016

Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

Cell Host Microbe 2016 05 21;19(5):675-85. Epub 2016 Apr 21.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2016.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867121PMC
May 2016

Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.

Proc Natl Acad Sci U S A 2015 Dec 14;112(52):E7249-56. Epub 2015 Dec 14.

Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710

Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1516305112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703010PMC
December 2015

Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer.

Proc Natl Acad Sci U S A 2015 Dec 30;112(50):E6945-54. Epub 2015 Nov 30.

Department of Molecular Genetics & Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710

Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus-but not poliovirus-replication in human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1513421112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687602PMC
December 2015

Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.

Genome Biol 2015 Nov 24;16:257. Epub 2015 Nov 24.

Editas Medicine, Cambridge, MA, 02142, USA.

Background: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9.

Results: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq.

Conclusion: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-015-0817-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657203PMC
November 2015

Targeting hepatitis B virus cccDNA using CRISPR/Cas9.

Antiviral Res 2015 Nov 22;123:188-92. Epub 2015 Oct 22.

Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, USA.

Despite the existence of an excellent prophylactic vaccine and the development of highly effective inhibitors of the viral polymerase, chronic hepatitis B virus (HBV) infection remains a major source of morbidity and mortality, especially in Africa and Asia. A significant problem is that, while polymerase inhibitors can effectively prevent the production of viral genomic DNA from pre-genomic RNA transcripts, they do not prevent the transcription and translation of viral mRNAs from the covalently closed circular DNA (cccDNA) templates present in the nuclei of infected cells. Moreover, because these cccDNAs are highly stable, chronic HBV infections are only very rarely cured by the use of polymerase inhibitors and these drugs clearly cannot entirely prevent the subsequent development of HBV-related morbidities such as cirrhosis and hepatocellular carcinoma. As a result, there has been considerable interest in the possibility of developing treatment approaches that directly target cccDNA for elimination. Here, we discuss recent publications that analyze the ability of the bacterial CRISPR/Cas DNA editing machinery to be repurposed as a tool for the specific cleavage and destruction of HBV cccDNAs in the nuclei of infected cells and consider which steps will be necessary to make CRISPR/Cas targeting of HBV DNA a clinically feasible approach to the treatment of chronic infections in humans. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2015.10.004DOI Listing
November 2015

Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

Methods 2015 Dec 17;91:82-86. Epub 2015 Aug 17.

Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States. Electronic address:

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2015.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684739PMC
December 2015

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

RNA 2015 Sep 17;21(9):1683-9. Epub 2015 Jul 17.

Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.051631.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536327PMC
September 2015

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

Virology 2015 May 7;479-480:213-20. Epub 2015 Mar 7.

Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, USA. Electronic address:

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes might be destroyed. In conclusion, we believe that the continued rapid evolution of CRISPR/Cas technology will soon have a major, possibly revolutionary, impact on the field of virology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2015.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424069PMC
May 2015

Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template.

Antiviral Res 2015 Mar 31;115:75-82. Epub 2014 Dec 31.

Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. Electronic address:

Non-dividing macrophages maintain extremely low cellular deoxyribonucleotide triphosphate (dNTP) levels, but high ribonucleotide triphosphate (rNTP) concentrations. The disparate nucleotide pools kinetically forces Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase (RT) to incorporate non-canonical rNTPs during reverse transcription. HIV-1 RT pauses near ribonucleoside monophosphates (rNMPs) embedded in the template DNA, which has previously been shown to enhance mismatch extension. Here, pre-steady state kinetic analysis shows rNTP binding affinity (Kd) of HIV-1 RT for non-canonical rNTPs was 1.4- to 43-fold lower, and the rNTP rate of incorporation (kpol) was 15- to 1551-fold slower than for dNTPs. This suggests that RT is more selective for incorporation of dNTPs rather than rNTPs. HIV-1 RT selectivity for dNTP versus rNTP is the lowest for ATP, implying that HIV-1 RT preferentially incorporates ATP when dATP concentration is limited. We observed that incorporation of a dNTP occurring one nucleotide before an embedded rNMP in the template had a 29-fold greater Kd and a 20-fold slower kpol as compared to the same template containing dNMP. This reduced the overall dNTP incorporation efficiency of HIV-1 RT by 581-fold. Finally, the RT mutant Y115F displayed lower discrimination against rNTPs due to its increase in binding affinity for non-canonical rNTPs. Overall, these kinetic results demonstrate that HIV-1 RT utilizes both substrate binding and a conformational change during: (1) enzymatic discrimination of non-canonical rNTPs from dNTPs and (2) during dNTP primer extension with DNA templates containing embedded rNMP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2014.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323949PMC
March 2015

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease.

Virology 2015 Feb 29;476:196-205. Epub 2014 Dec 29.

Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, United States. Electronic address:

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2014.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323668PMC
February 2015

Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease.

J Virol 2014 Oct 6;88(20):11965-72. Epub 2014 Aug 6.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA

High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01879-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178730PMC
October 2014
-->