Brett Lindenbach, PhD
Research & Publications
Biography
News
Research Summary
Research in the Lindenbach laboratory focuses on the replication of enveloped, positive-strand RNA viruses, including flaviviruses (yellow fever virus, dengue virus, Zika virus) and hepatitis C virus (HCV). Specifically, we combine genetic, biochemical, and cell biological approaches to study how viral structural and nonstructural (NS) proteins contribute to viral genome replication and to the assembly of infectious particles. We have also developed novel methods to reveal essential interactions between viruses and host cells.
Extensive Research Description
Many RNA viruses encode RNA helicases that are essential for viral replication, and it is frequently assumed that these enzymes unwind double-stranded forms of the viral gemone. Indeed, many of these enzymes have been shown to have RNA binding, ATPase, and RNA unwinding activities in vitro. However, to date there is no direct evidence that these enzymes bind to or unwind viral RNA in infected cells. We recently identified several important activities of the HCV NS3-4A RNA helicase domain in recruiting RNA an template for replication and in in virus assembly.
RNA replication
For many HCV nonstructural (NS) proteins, biochemical activities have been characterized and several high-resolution crystal structures are available. However what we most lack is an understanding of how these pieces work together to form the active replication complex, and how host cofactors influence the steps of translation and replication. We are combining genetic and biochemical approaches to close this gap in our knowledge. Specifically, we have developed a novel trans-complementation system to dissect the features of viral NS proteins proteins required for assembly of functional replication complexes. By using this system, we discovered the HCV NS3-4A helicase recruits the viral genome in cis (i.e., the same RNA from which it is translated) out of translation and into RNA replication. We also found that NS5B has an essential cis-acting role in RNA replication, independent of its RNA binding and RNA polymerase activities. 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.
Virus assembly
We discovered that the HCV NS2 protein interacts with both the viral E1-E2 glycoprotein complex and the NS3-4A enzyme complex and that these interactions are essential for virus particle assembly. To examine the cell biology of HCV particle assembly in greater detail, we developed methods to fluorescently label functional core protein in virus-producing cells. Our data revealed that core protein is rapidly trafficked to the surface of lipid droplets, which associate with the sites of virus assembly at the ER. After egress from lipid droplets, core protein is incorporated into virus particles, which bud into the ER and traffic via the secretory pathway. By examining core trafficking in NS2 mutants with or without second-site genetic suppressors in NS3, we showed that the interaction between NS2 and NS3-4A is essential for recruiting core from the surface of lipid droplets into virus particles. Our current working model is that the interaction between NS2 and NS3-4A regulates the flow of RNA out of replication and into packaging. Because RNA helicase activity is essential for RNA replication, which is a pre-requisite for virus assembly, we have developed a unqiue genetic approach to separate the functions of the NS3 helicase in viral genome replication from its role in virus assembly.
Bacterial effectors as probes to study (+) RNA virus-host cell biology
The mechanisms by which viruses interact with their host cells are incompletely understood; identifying these interactions remains a fundamentally important area of basic virus research. The three most common approaches to discovering virus-host cell interaction have been: 1) Genome-wide RNAi screens for host genes that influence viral replication; 2) Identifying protein-protein interactions via proteomics or genetic two-hybrid screens; 3) Screening pharmacological agents to disrupt known cellular pathways. While these approaches have been incredibly useful, their limitations include: variability in RNAi knockdown efficiency, off-target effects, limited reproducibility between genome-wide screens, false-positive scoring of protein-protein interactions, and a relatively small and nonspecific pharmacopeia. We are exploring a new strategy to identify virus-host cell interaction by employing a large collection of bacterial effector proteins as a genetic toolkit to surgically manipulate key cellular pathways. Many bacterial pathogens infect and survive within eukaryotic cells by injecting minute quantities of bacterial effector proteins, typically enzymes, into the cytosol of their hosts. These effector proteins have evolved to manipulate cellular pathways, prevent bacterial degradation, and favor bacterial replication. For instance, Legionella pneumophila, the causative agent of Legionnaire’s disease, synthesizes >300 effector proteins, some of which reprogram endolysosomal membrane trafficking, potently inhibit cellular autophagy, and divert innate immune responses. Importantly, many effector proteins retain their function when ectopically expressed in mammalian cells and can be used to study cellular pathways independent of bacterial infection. Bacterial effector proteins frequently target the same cellular pathways used by (+) RNA viruses. For instance, Legionella effector proteins manipulate Rab1, a key organizer of ER-to- Golgi membrane traffic and a host factor required for hepatitis C virus (HCV) replication. Other effectors inhibit autophagy, a pathway exploited by HCV and many other (+) RNA viruses. Based on these known functional overlaps we hypothesize that bacterial effector proteins can be used as tools to identify cellular pathways used by (+) RNA viruses.
Coauthors
Research Interests
Arbovirus Infections; Biochemistry; Biology; Biotechnology; Genetic Techniques; Hepatitis, Viral, Human; Hepatitis C; Liver Diseases; Microscopy; RNA; RNA Virus Infections; Viruses
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Selected Publications
- Reinventing positive-strand RNA virus reverse geneticsLindenbach BD. Reinventing positive-strand RNA virus reverse genetics. 2022, 112: 1-29. PMID: 35840179, PMCID: PMC9273853, DOI: 10.1016/bs.aivir.2022.03.001.
- The In Vivo and In Vitro Architecture of the Hepatitis C Virus RNA Genome Uncovers Functional RNA Secondary and Tertiary StructuresWan H, Adams RL, Lindenbach BD, Pyle AM. The In Vivo and In Vitro Architecture of the Hepatitis C Virus RNA Genome Uncovers Functional RNA Secondary and Tertiary Structures. Journal Of Virology 2022, 96: e01946-21. PMID: 35353000, PMCID: PMC9044954, DOI: 10.1128/jvi.01946-21.
- De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case reportGandhi S, Klein J, Robertson AJ, Peña-Hernández MA, Lin MJ, Roychoudhury P, Lu P, Fournier J, Ferguson D, Mohamed Bakhash SAK, Catherine Muenker M, Srivathsan A, Wunder EA, Kerantzas N, Wang W, Lindenbach B, Pyle A, Wilen CB, Ogbuagu O, Greninger AL, Iwasaki A, Schulz WL, Ko AI. De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case report. Nature Communications 2022, 13: 1547. PMID: 35301314, PMCID: PMC8930970, DOI: 10.1038/s41467-022-29104-y.
- De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: A case report.Gandhi S, Klein J, Robertson A, Peña-Hernández MA, Lin MJ, Roychoudhury P, Lu P, Fournier J, Ferguson D, Mohamed Bakhash SA, Catherine Muenker M, Srivathsan A, Wunder EA, Kerantzas N, Wang W, Lindenbach B, Pyle A, Wilen CB, Ogbuagu O, Greninger AL, Iwasaki A, Schulz WL, Ko AI. De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: A case report. MedRxiv : The Preprint Server For Health Sciences 2021 PMID: 34909781, PMCID: PMC8669848, DOI: 10.1101/2021.11.08.21266069.
- Placenta‐derived interferon‐stimulated gene 20 controls ZIKA virus infectionDing J, Aldo P, Roberts CM, Stabach P, Liu H, You Y, Qiu X, Jeong J, Maxwell A, Lindenbach B, Braddock D, Liao A, Mor G. Placenta‐derived interferon‐stimulated gene 20 controls ZIKA virus infection. EMBO Reports 2021, 22: e52450. PMID: 34405956, PMCID: PMC8490983, DOI: 10.15252/embr.202152450.
- Optimization of Triarylpyridinone Inhibitors of the Main Protease of SARS-CoV‑2 to Low-Nanomolar Antiviral PotencyZhang CH, Spasov KA, Reilly RA, Hollander K, Stone EA, Ippolito JA, Liosi ME, Deshmukh MG, Tirado-Rives J, Zhang S, Liang Z, Miller SJ, Isaacs F, Lindenbach BD, Anderson KS, Jorgensen WL. Optimization of Triarylpyridinone Inhibitors of the Main Protease of SARS-CoV‑2 to Low-Nanomolar Antiviral Potency. ACS Medicinal Chemistry Letters 2021, 12: 1325-1332. PMID: 34408808, PMCID: PMC8291137, DOI: 10.1021/acsmedchemlett.1c00326.
- Restriction of SARS-CoV-2 replication by targeting programmed −1 ribosomal frameshiftingSun Y, Abriola L, Niederer RO, Pedersen SF, Alfajaro MM, Silva Monteiro V, Wilen CB, Ho YC, Gilbert WV, Surovtseva YV, Lindenbach BD, Guo JU. Restriction of SARS-CoV-2 replication by targeting programmed −1 ribosomal frameshifting. Proceedings Of The National Academy Of Sciences Of The United States Of America 2021, 118: e2023051118. PMID: 34185680, PMCID: PMC8256030, DOI: 10.1073/pnas.2023051118.
- Potent Noncovalent Inhibitors of the Main Protease of SARS-CoV‑2 from Molecular Sculpting of the Drug Perampanel Guided by Free Energy Perturbation CalculationsZhang CH, Stone EA, Deshmukh M, Ippolito JA, Ghahremanpour MM, Tirado-Rives J, Spasov KA, Zhang S, Takeo Y, Kudalkar SN, Liang Z, Isaacs F, Lindenbach B, Miller SJ, Anderson KS, Jorgensen WL. Potent Noncovalent Inhibitors of the Main Protease of SARS-CoV‑2 from Molecular Sculpting of the Drug Perampanel Guided by Free Energy Perturbation Calculations. ACS Central Science 2021, 7: 467-475. PMID: 33786375, PMCID: PMC7931627, DOI: 10.1021/acscentsci.1c00039.
- Enabling HCV replication in cell culture: An approach for vaccine and drug developmentNgounoue M, Ndjomou J, Lindenbach B. Enabling HCV replication in cell culture: An approach for vaccine and drug development. International Journal Of Infectious Diseases 2020, 101: 510. DOI: 10.1016/j.ijid.2020.09.1325.
- Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro.Sun Y, Abriola L, Surovtseva YV, Lindenbach BD, Guo JU. Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro. BioRxiv : The Preprint Server For Biology 2020 PMID: 33106809, PMCID: PMC7587830, DOI: 10.1101/2020.10.21.349225.
- A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus UncoatingZhang S, Ramanathan H, Douam F, Mar K, Chang J, Yang P, Schoggins J, Ploss A, Lindenbach B. A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating. 2020, 147. DOI: 10.3390/proceedings2020050147.
- Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial GliaOnorati M, Li Z, Liu F, Sousa AMM, Nakagawa N, Li M, Dell’Anno M, Gulden FO, Pochareddy S, Tebbenkamp AT, Han W, Pletikos M, Gao T, Zhu Y, Bichsel C, Varela L, Szigeti-Buck K, Lisgo S, Zhang Y, Testen A, Gao XB, Mlakar J, Popovic M, Flamand M, Strittmatter SM, Kaczmarek LK, Anton ES, Horvath TL, Lindenbach BD, Sestan N. Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia. Cell Reports 2016, 16: 2576-2592. PMID: 27568284, PMCID: PMC5135012, DOI: 10.1016/j.celrep.2016.08.038.
- Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain InfectionYockey LJ, Varela L, Rakib T, Khoury-Hanold W, Fink SL, Stutz B, Szigeti-Buck K, Van den Pol A, Lindenbach BD, Horvath TL, Iwasaki A. Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection. Cell 2016, 166: 1247-1256.e4. PMID: 27565347, PMCID: PMC5006689, DOI: 10.1016/j.cell.2016.08.004.
- The Coding Region of the HCV Genome Contains a Network of Regulatory RNA StructuresPirakitikulr N, Kohlway A, Lindenbach BD, Pyle AM. The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures. Molecular Cell 2016, 62: 111-120. PMID: 26924328, PMCID: PMC4826301, DOI: 10.1016/j.molcel.2016.01.024.
- Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural ProteinsKazakov T, Yang F, Ramanathan HN, Kohlway A, Diamond MS, Lindenbach BD. Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins. PLOS Pathogens 2015, 11: e1004817. PMID: 25875808, PMCID: PMC4395149, DOI: 10.1371/journal.ppat.1004817.
- Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane DomainKohlway A, Pirakitikulr N, Barrera FN, Potapova O, Engelman DM, Pyle AM, Lindenbach BD. Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain. Journal Of Virology 2013, 88: 628-642. PMID: 24173222, PMCID: PMC3911751, DOI: 10.1128/jvi.02052-13.
- The ins and outs of hepatitis C virus entry and assemblyLindenbach BD, Rice CM. The ins and outs of hepatitis C virus entry and assembly. Nature Reviews Microbiology 2013, 11: 688-700. PMID: 24018384, PMCID: PMC3897199, DOI: 10.1038/nrmicro3098.
- Flaviviridae: the viruses and their replication.Lindenbach, B.D., Murray, C.L., Thiel, H.-J., Rice, C.M. (2007) Fields Virology, 5th Ed.
- Hepatitis C Virus and Hepatocellular CarcinomaLindenbach B. Hepatitis C Virus and Hepatocellular Carcinoma. 2011, 571-583. DOI: 10.1007/978-1-4614-0016-5_23.
- Trafficking of Hepatitis C Virus Core Protein during Virus Particle AssemblyCounihan NA, Rawlinson SM, Lindenbach BD. Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly. PLOS Pathogens 2011, 7: e1002302. PMID: 22028650, PMCID: PMC3197604, DOI: 10.1371/journal.ppat.1002302.
- Structural Insights into RNA Recognition by RIG-ILuo D, Ding SC, Vela A, Kohlway A, Lindenbach BD, Pyle AM. Structural Insights into RNA Recognition by RIG-I. Cell 2011, 147: 409-422. PMID: 22000018, PMCID: PMC3222294, DOI: 10.1016/j.cell.2011.09.023.
- Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme ComplexesStapleford KA, Lindenbach BD. Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes. Journal Of Virology 2010, 85: 1706-1717. PMID: 21147927, PMCID: PMC3028914, DOI: 10.1128/jvi.02268-10.
- The Acidic Domain of Hepatitis C Virus NS4A Contributes to RNA Replication and Virus Particle AssemblyPhan T, Kohlway A, Dimberu P, Pyle AM, Lindenbach BD. The Acidic Domain of Hepatitis C Virus NS4A Contributes to RNA Replication and Virus Particle Assembly. Journal Of Virology 2010, 85: 1193-1204. PMID: 21047963, PMCID: PMC3020511, DOI: 10.1128/jvi.01889-10.
- Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes ▿Phan T, Beran RK, Peters C, Lorenz IC, Lindenbach BD. Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes ▿. Journal Of Virology 2009, 83: 8379-8395. PMID: 19515772, PMCID: PMC2738163, DOI: 10.1128/jvi.00891-09.
- Hepatitis C Virus Genome ReplicationLindenbach B, L. Tellinghuisen T. Hepatitis C Virus Genome Replication. 2009, 61-88. DOI: 10.1007/b135974_4.
- Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitroLindenbach BD, Meuleman P, Ploss A, Vanwolleghem T, Syder AJ, McKeating JA, Lanford RE, Feinstone SM, Major ME, Leroux-Roels G, Rice CM. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proceedings Of The National Academy Of Sciences Of The United States Of America 2006, 103: 3805-3809. PMID: 16484368, PMCID: PMC1533780, DOI: 10.1073/pnas.0511218103.
- Investigations into the receptor requirements of Hepatitis C Virus (HCV) pseudoparticles and cell culture grown virionsvon Hahn T, Flint M, Lindenbach B, Boullier A, Quehenberger O, Rice C, McKeating J. Investigations into the receptor requirements of Hepatitis C Virus (HCV) pseudoparticles and cell culture grown virions. Zeitschrift Für Gastroenterologie 2006, 44 DOI: 10.1055/s-2006-931763.
- Unravelling hepatitis C virus replication from genome to functionLindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function. Nature 2005, 436: 933-938. PMID: 16107832, DOI: 10.1038/nature04077.
- Complete Replication of Hepatitis C Virus in Cell CultureLindenbach BD, Evans MJ, Syder AJ, Wölk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM. Complete Replication of Hepatitis C Virus in Cell Culture. Science 2005, 309: 623-626. PMID: 15947137, DOI: 10.1126/science.1114016.
- 381 Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes in living cellsMoradpour D, Evans M, Gosert R, Yuan Z, Blum H, Goff S, Lindenbach B, Rice C. 381 Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes in living cells. Hepatology 2003, 38: 343. DOI: 10.1016/s0270-9139(03)80424-x.
- Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function.Lindenbach B, Rice C. Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. Journal Of Virology 1999, 73: 4611-21. PMID: 10233920, PMCID: PMC112502, DOI: 10.1128/jvi.73.6.4611-4621.1999.
- Noncytopathic Sindbis virus RNA vectors for heterologous gene expressionAgapov E, Frolov I, Lindenbach B, Prágai B, Schlesinger S, Rice C. Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proceedings Of The National Academy Of Sciences Of The United States Of America 1998, 95: 12989-12994. PMID: 9789028, PMCID: PMC23682, DOI: 10.1073/pnas.95.22.12989.
- Construction of Recombinant Sindbis-Based Expression Vectors for the Study of HCV Genes and Their ProductsLindenbach B, Frolov I, Rice C. Construction of Recombinant Sindbis-Based Expression Vectors for the Study of HCV Genes and Their Products. 1998, 19: 565-574. PMID: 21374397, DOI: 10.1385/0-89603-521-2:565.
- trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication.Lindenbach B, Rice C. trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. Journal Of Virology 1997, 71: 9608-17. PMID: 9371625, PMCID: PMC230269, DOI: 10.1128/jvi.71.12.9608-9617.1997.
- Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini.Lin C, Lindenbach B, Prágai B, McCourt D, Rice C. Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. Journal Of Virology 1994, 68: 5063-73. PMID: 7518529, PMCID: PMC236449, DOI: 10.1128/jvi.68.8.5063-5073.1994.