Publications by authors named "Brett D Lindenbach"

59 Publications

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro.

bioRxiv 2020 Oct 21. Epub 2020 Oct 21.

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed -1 ribosomal frameshifting (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting -1 PRF as an effective antiviral strategy for SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.10.21.349225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587830PMC
October 2020

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.

J Biol Chem 2020 08 8;295(35):12426-12436. Epub 2020 Jul 8.

Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* and We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys and Thr and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.
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http://dx.doi.org/10.1074/jbc.RA120.013455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458815PMC
August 2020

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating.

mBio 2020 04 14;11(2). Epub 2020 Apr 14.

Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, USA

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
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http://dx.doi.org/10.1128/mBio.00467-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157815PMC
April 2020

Screening effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication.

Proc Natl Acad Sci U S A 2019 10 7;116(43):21739-21747. Epub 2019 Oct 7.

Department of Plant Pathology, University of Kentucky, Lexington, KY 40546;

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our effector screen, we used the effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.
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http://dx.doi.org/10.1073/pnas.1911108116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815150PMC
October 2019

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment.

Cell Rep 2019 02;26(7):1800-1814.e5

Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.
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http://dx.doi.org/10.1016/j.celrep.2019.01.075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053169PMC
February 2019

Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release.

mBio 2018 03 13;9(2). Epub 2018 Mar 13.

Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread. HCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence. Yet, factors governing this differential sorting remain unknown. By integrating proteomic, RNA interference, genetic, live-cell imaging, and pharmacological approaches, we uncover differential coopting of host adaptor proteins (APs) to mediate HCV traffic at distinct late steps of the viral life cycle. We reported that AP-1A and AP-2 mediate HCV trafficking during release and assembly, respectively. Here, we demonstrate that dileucine motifs in the NS2 protein mediate AP-1A, AP-1B, and AP-4 binding and cell-free virus release. Moreover, we reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. Lastly, we demonstrate cell-to-cell spread regulation by AAK1 and GAK, host kinases controlling APs, and susceptibility to their inhibitors. This study provides mechanistic insights into virus-host determinants that facilitate HCV trafficking, with potential implications for pathogenesis and antiviral agent design.
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http://dx.doi.org/10.1128/mBio.02233-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850324PMC
March 2018

Critical challenges and emerging opportunities in hepatitis C virus research in an era of potent antiviral therapy: Considerations for scientists and funding agencies.

Virus Res 2018 03 2;248:53-62. Epub 2018 Mar 2.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

The development and clinical implementation of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C. Infection with any hepatitis C virus (HCV) genotype can now be eliminated in more than 95% of patients with short courses of all-oral, well-tolerated drugs, even in those with advanced liver disease and liver transplant recipients. DAAs have proven so successful that some now consider HCV amenable to eradication, and continued research on the virus of little remaining medical relevance. However, given 400,000 HCV-related deaths annually important challenges remain, including identifying those who are infected, providing access to treatment and reducing its costs. Moreover, HCV infection rarely induces sterilizing immunity, and those who have been cured with DAAs remain at risk for reinfection. Thus, it is very unlikely that global eradication and elimination of the cancer risk associated with HCV infection can be achieved without a vaccine, yet research in that direction receives little attention. Further, over the past two decades HCV research has spearheaded numerous fundamental discoveries in the fields of molecular and cell biology, immunology and microbiology. It will continue to do so, given the unique opportunities afforded by the reagents and knowledge base that have been generated in the development and clinical application of DAAs. Considering these critical challenges and new opportunities, we conclude that funding for HCV research must be sustained.
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http://dx.doi.org/10.1016/j.virusres.2018.02.016DOI Listing
March 2018

Recovery of Infectious Hepatitis C Virus From Injection Paraphernalia: Implications for Prevention Programs Serving People Who Inject Drugs.

J Infect Dis 2018 01;217(3):466-473

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut.

Background: Controlling hepatitis C virus (HCV) transmission among people who inject drugs (PWID) has focused on preventing sharing syringes and drug preparation paraphernalia, but it is unclear whether HCV incidence linked to sharing paraphernalia reflects contamination of the paraphernalia or syringe-mediated contamination when drugs are shared.

Methods: In experiments designed to replicate real-world injection practices when drugs are shared, the residual contents of HCV-contaminated syringes with detachable or fixed needled were passed through the "cookers" and filters used by PWID in preparing drugs for injection and then introduced into a second syringe. All items were tested for the presence of infectious HCV using a chimeric HCV with a luciferase gene.

Results: Hepatitis C virus could not be recovered from cookers regardless of input syringe type or cooker design. Recovery was higher when comparing detachable needles to fixed needles for residue in input syringes (73.8% vs 0%), filters (15.4% vs 1.4%), and receptive syringes (93.8% vs 45.7%).

Conclusions: Our results, consistent with the hypothesis that sharing paraphernalia does not directly result in HCV transmission but is a surrogate for transmissions resulting from sharing drugs, have important implications for HCV prevention efforts and programs that provide education and safe injection supplies for PWID populations.
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http://dx.doi.org/10.1093/infdis/jix427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854006PMC
January 2018

IRE1α promotes viral infection by conferring resistance to apoptosis.

Sci Signal 2017 Jun 6;10(482). Epub 2017 Jun 6.

Department of Immunobiology, Yale University, New Haven, CT 06520, USA.

The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-folding stresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) promote type I interferon (IFN) responses. We found that -deficient mouse embryonic fibroblasts and macrophages had impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather an inability of -deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infection in wild-type cells. -deficient cells were generally resistant to the intrinsic pathway of apoptosis through an indirect mechanism involving activation of the nuclease IRE1α. We observed an IRE1α-dependent reduction in the abundance of the proapoptotic microRNA miR-125a and a corresponding increase in the amounts of the members of the antiapoptotic Bcl-2 family. The activation of IRE1α by the hepatitis C virus (HCV) protein NS4B in XBP1-proficient cells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1α activation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to those in the livers of healthy controls. Our results reveal a prosurvival role for IRE1α in virally infected cells and suggest a possible target for IFN-independent antiviral therapy.
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http://dx.doi.org/10.1126/scisignal.aai7814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535312PMC
June 2017

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

PLoS Negl Trop Dis 2016 Oct 5;10(10):e0005048. Epub 2016 Oct 5.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions.

Methodology/principal Findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action.

Conclusions/significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.
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http://dx.doi.org/10.1371/journal.pntd.0005048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5051680PMC
October 2016

Zika virus productively infects primary human placenta-specific macrophages.

JCI Insight 2016 Aug;1(13)

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus-associated pregnancy complications.
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http://dx.doi.org/10.1172/jci.insight.88461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007065PMC
August 2016

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

Cell Rep 2016 09 24;16(10):2576-2592. Epub 2016 Aug 24.

Department of Neuroscience and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Cellular Neuroscience, Neurodegeneration and Repair Program, Departments of Neurology and Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Section of Comparative Medicine, Yale School of Medicine, New Haven, CT 06510, USA; Departments of Genetics and Psychiatry, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address:

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.
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http://dx.doi.org/10.1016/j.celrep.2016.08.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135012PMC
September 2016

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

Cell 2016 Aug;166(5):1247-1256.e4

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520 USA; Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06520 USA. Electronic address:

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.
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http://dx.doi.org/10.1016/j.cell.2016.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006689PMC
August 2016

The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures.

Mol Cell 2016 Apr 25;62(1):111-20. Epub 2016 Feb 25.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA; Department of Chemistry, Yale University, New Haven, CT 06511, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

RNA is a versatile macromolecule that accommodates functional information in primary sequence and secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, comparative sequence analysis, and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus' life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a "code within the code" for controlling biological processes.
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http://dx.doi.org/10.1016/j.molcel.2016.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826301PMC
April 2016

What's next for hepatitis C virus research?

Hepatology 2016 May 19;63(5):1408-10. Epub 2016 Mar 19.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT.

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http://dx.doi.org/10.1002/hep.28494DOI Listing
May 2016

Survival of Hepatitis C Virus in Syringes Is Dependent on the Design of the Syringe-Needle and Dead Space Volume.

PLoS One 2015 4;10(11):e0139737. Epub 2015 Nov 4.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Background: Many people who inject drugs (PWID) use syringes with detachable needles, which have high dead space (HDS). Contaminated HDS blood may substantially contribute to the transmission of HIV, hepatitis C (HCV), and other blood-borne viruses within this population. Newly designed low dead space (LDS) syringe-needle combinations seek to reduce blood-borne virus transmission among PWID. We evaluated the infectivity of HCV-contaminated residual volumes recovered from two LDS syringe-needle combinations.

Methods: We tested two different design approaches to reducing the dead space. One added a piston to the plunger; the other reduced the dead space within the needle. The two approaches cannot be combined. Recovery of genotype-2a reporter HCV from LDS syringe-needle combinations was compared to recovery from insulin syringes with fixed needles and standard HDS syringe-needle combinations. Recovery of HCV from syringes was determined immediately following their contamination with HCV-spiked plasma, after storage at 22°C for up to 1 week, or after rinsing with water.

Results: Insulin syringes with fixed needles had the lowest proportion of HCV-positive syringes before and after storage. HCV recovery after immediate use ranged from 47%±4% HCV-positive 1 mL insulin syringes with 27-gauge ½ inch needles to 98%±1% HCV-positive HDS 2 mL syringes with 23-gauge 1¼ inch detachable needles. LDS combinations yielded recoveries ranging from 65%±5% to 93%±3%. Recovery was lower in combinations containing LDS needles than LDS syringes. After 3 days of storage, as much as 6-fold differences in virus recovery was observed, with HCV recovery being lower in combinations containing LDS needles. Most combinations with detachable needles required multiple rinses to reduce HCV infectivity to undetectable levels whereas a single rinse of insulin syringes was sufficient.

Conclusions: Our study, the first to assess the infectivity of HCV in residual volumes of LDS syringes and needles available to PWID, demonstrates that LDS syringe-needle combination still has the greater potential for HCV transmission than insulin syringes with fixed needles. Improved LDS designs may be able to further reduce HCV recovery, but based on the designed tested, LDS needles and syringes remain intermediate between fixed-needle syringes and HDS combinations in reducing exposure to HCV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139737PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633215PMC
June 2016

Disinfection of syringes contaminated with hepatitis C virus by rinsing with household products.

Open Forum Infect Dis 2015 Jan 23;2(1):ofv017. Epub 2015 Feb 23.

Department of Epidemiology of Microbial Diseases , Yale School of Public Health , New Haven, Connecticut.

Background.  Hepatitis C virus (HCV) transmission among people who inject drugs (PWID) is associated with the sharing of injection paraphernalia. People who inject drugs often "disinfect" used syringes with household products when new syringes are unavailable. We assessed the effectiveness of these products in disinfecting HCV-contaminated syringes. Methods.  A genotype-2a reporter virus assay was used to assess HCV infectivity in syringes postrinsing. Hepatitis C virus-contaminated 1 mL insulin syringes with fixed needles and 1 mL tuberculin syringes with detachable needles were rinsed with water, Clorox bleach, hydrogen peroxide, ethanol, isopropanol, Lysol, or Dawn Ultra at different concentrations. Syringes were either immediately tested for viable virus or stored at 4°C, 22°C, and 37°C for up to 21 days before viral infectivity was determined. Results.  Most products tested reduced HCV infectivity to undetectable levels in insulin syringes. Bleach eliminated HCV infectivity in both syringes. Other disinfectants produced virus recovery ranging from high (5% ethanol, 77% ± 12% HCV-positive syringes) to low (1:800 Dawn Ultra, 7% ± 7% positive syringes) in tuberculin syringes. Conclusions.  Household disinfectants tested were more effective in fixed-needle syringes (low residual volume) than in syringes with detachable needles (high residual volume). Bleach was the most effective disinfectant after 1 rinse, whereas other diluted household products required multiple rinses to eliminate HCV. Rinsing with water, 5% ethanol (as in beer), and 20% ethanol (as in fortified wine) was ineffective and should be avoided. Our data suggest that rinsing of syringes with household disinfectants may be an effective tool in preventing HCV transmission in PWID when done properly.
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http://dx.doi.org/10.1093/ofid/ofv017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438897PMC
January 2015

Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins.

PLoS Pathog 2015 Apr 13;11(4):e1004817. Epub 2015 Apr 13.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.
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http://dx.doi.org/10.1371/journal.ppat.1004817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395149PMC
April 2015

A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication.

PLoS Pathog 2015 Mar 16;11(3):e1004736. Epub 2015 Mar 16.

Institute of Virology and Cell Biology, University of Lübeck, Germany.

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.
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http://dx.doi.org/10.1371/journal.ppat.1004736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361677PMC
March 2015

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

J Biol Chem 2014 Aug 16;289(35):24397-416. Epub 2014 Jul 16.

From the Department of Biochemistry and Molecular Biology,

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.
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http://dx.doi.org/10.1074/jbc.M114.589911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148867PMC
August 2014

The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus.

J Virol 2014 Sep 25;88(18):10970-4. Epub 2014 Jun 25.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA Department of Chemistry, Yale University, New Haven, Connecticut, USA Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro. Thus, we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than for modulating the enzymatic functions of NS3-4A.
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http://dx.doi.org/10.1128/JVI.00745-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178846PMC
September 2014

Hepatitis C virus maintains infectivity for weeks after drying on inanimate surfaces at room temperature: implications for risks of transmission.

J Infect Dis 2014 Apr 23;209(8):1205-11. Epub 2013 Nov 23.

Departments of Pediatrics and Pharmacology, Yale School of Medicine.

Background: Healthcare workers may come into contact with fomites that contain infectious hepatitis C virus (HCV) during preparation of plasma or following placement or removal of venous lines. Similarly, injection drugs users may come into contact with fomites. Hypothesizing that prolonged viability of HCV in fomites may contribute significantly to incidence, we determined the longevity of virus infectivity and the effectiveness of antiseptics.

Methods: We determined the volume of drops misplaced during transfer of serum or plasma. Aliquots equivalent to the maximum drop volume of plasma spiked with the 2a HCV reporter virus were loaded into 24-well plates. Plates were stored uncovered at 3 temperatures: 4°C, 22°C, and 37°C for up to 6 weeks before viral infectivity was determined in a microculture assay.

Results: The mean volume of an accidental drop was 29 µL (min-max of 20-33 µL). At storage temperatures 4°C and 22°C, we recovered viable HCV from the low-titer spots for up to 6 weeks of storage. The rank order of HCV virucidal activity of commonly used antiseptics was bleach (1:10) > cavicide (1:10) > ethanol (70%).

Conclusions: The hypothesis of potential transmission from fomites was supported by the experimental results. The anti-HCV activity of commercial antiseptics varied.
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http://dx.doi.org/10.1093/infdis/jit648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3969546PMC
April 2014

Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain.

J Virol 2014 Jan 30;88(1):628-42. Epub 2013 Oct 30.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.
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http://dx.doi.org/10.1128/JVI.02052-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911751PMC
January 2014

The ins and outs of hepatitis C virus entry and assembly.

Nat Rev Microbiol 2013 Oct 10;11(10):688-700. Epub 2013 Sep 10.

Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut 06536, USA.

Hepatitis C virus, a major human pathogen, produces infectious virus particles with several unique features, such as an ability to interact with serum lipoproteins, a dizzyingly complicated process of virus entry, and a pathway of virus assembly and release that is closely linked to lipoprotein secretion. Here, we review these unique features, with an emphasis on recent discoveries concerning virus particle structure, virus entry and virus particle assembly and release.
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http://dx.doi.org/10.1038/nrmicro3098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897199PMC
October 2013

Virion assembly and release.

Curr Top Microbiol Immunol 2013 ;369:199-218

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA.

Hepatitis C Virus (HCV) particles exhibit several unusual properties that are not found in other enveloped RNA viruses, most notably their low buoyant density and interaction with serum lipoproteins. With the advent of systems to grow HCV in cell culture, the molecular basis of HCV particle assembly and release can now be addressed. The process of virus assembly involves protein-protein interactions between viral structural and nonstructural proteins and the coordinated action of host factors. This chapter reviews our current understanding of these interactions and factors.
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http://dx.doi.org/10.1007/978-3-642-27340-7_8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925669PMC
August 2013

Trafficking of hepatitis C virus core protein during virus particle assembly.

PLoS Pathog 2011 Oct 20;7(10):e1002302. Epub 2011 Oct 20.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.
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http://dx.doi.org/10.1371/journal.ppat.1002302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197604PMC
October 2011