Arbovirus Infections; Biochemistry; Biology; Biotechnology; Genetic Techniques; Hepatitis, Viral, Human; Hepatitis C; Liver Diseases; Microscopy; RNA; RNA Virus Infections; Viruses
Molecular Virology: Virology laboratories
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.
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.
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.
Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins.
Kazakov T, Yang F, Ramanathan HN, Kohlway A, Diamond MS, Lindenbach BD (2015) PLoS Pathogens 11(4):e1004817.
Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain.
Kohlway A, Pirakitikulr N, Barrera FN, Potapova O, Engelman DM, Pyle AM, Lindenbach BD (2014) "." J. Virol. 88(1):628-42.
The ins and outs of hepatitis C virus entry and assembly.
Lindenbach BD, Rice CM. (2013) Nature Reviews Microbiology. 2013 Oct;11(10):688-700.
Structural Insights into RNA Recognition by RIG-I.
Luo D., Ding S.C., Vela A., Kohlway A., Lindenbach B.D., Pyle A.M. (2011). Cell 147(2):409-22.
Trafficking of Hepatitis C Virus Core Protein During Virus Particle Assembly.
Counihan N.A., Rawlinson S.M., Lindenbach B.D. (2011) PLoS Pathogens 7(10): e1002302.
Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.
Stapleford K.A., Lindenbach B.D. (2011) J Virol. 85(4):1706-17.
The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly.
Phan T., Kohlway A., Dimberu P., Pyle A.M., Lindenbach B.D. (2011) J Virol. 85(3):1193-204.
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 R.K., Peters C., Lorenz I.C., Lindenbach B.D. (2009). J. Virol. (83):8379-95.
Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.
Lindenbach, B.D., et al. (2006). Proc. Natl. Acad. Sci. (USA) 103:3805-3809.
Complete replication of hepatitis C virus in cell culture.
Lindenbach, B.D., et al. (2005). Science 309:623-626.
- Flaviviridae: the viruses and their replication. Lindenbach, B.D., Murray, C.L., Thiel, H.-J., Rice, C.M. (2007) Fields Virology, 5th Ed.
Unravelling hepatitis C virus replication from genome to function.
Lindenbach BD, Rice CM. (2005) Nature 436, 933-938.
The coding region of the HCV genome contains a network of regulatory RNA structures.
Pirakitikulr N. Kohlway A., Lindenbach B.D., and A.M. Pyle. Molecular Cell 2016, 62(1):111-20.
Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.
Onorati M, et al., Cell Rep. 2016 Aug 23. pii: S2211-1247
Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection
Yockey L.J., et al., Cell. 2016 Aug 25;166(5):1247-1256
Full List of PubMed Publications
- Aldo P, You Y, Szigeti K, Horvath TL, Lindenbach B, Mor G: HSV-2 enhances ZIKV infection of the placenta and induces apoptosis in first-trimester trophoblast cells. Am J Reprod Immunol. 2016 Nov; 2016 Sep 10. PMID: 27613665
- Barouch-Bentov R, Neveu G, Xiao F, Beer M, Bekerman E, Schor S, Campbell J, Boonyaratanakornkit J, Lindenbach B, Lu A, Jacob Y, Einav S: Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment. MBio. 2016 Nov 1; 2016 Nov 1. PMID: 27803188
- Donald CL, Brennan B, Cumberworth SL, Rezelj VV, Clark JJ, Cordeiro MT, Freitas de Oliveira França R, Pena LJ, Wilkie GS, Da Silva Filipe A, Davis C, Hughes J, Varjak M, Selinger M, Zuvanov L, Owsianka AM, Patel AH, McLauchlan J, Lindenbach BD, Fall G, Sall AA, Biek R, Rehwinkel J, Schnettler E, Kohl A: Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil. PLoS Negl Trop Dis. 2016 Oct; 2016 Oct 5. PMID: 27706161
- Onorati M, Li Z, Liu F, Sousa AM, Nakagawa N, Li M, Dell'Anno MT, 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 Rep. 2016 Sep 6; 2016 Aug 24. PMID: 27568284
- Yockey 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 Aug 25. PMID: 27565347
- Jurado KA, Simoni MK, Tang Z, Uraki R, Hwang J, Householder S, Wu M, Lindenbach BD, Abrahams VM, Guller S, Fikrig E: Zika virus productively infects primary human placenta-specific macrophages. JCI Insight. 2016 Aug 18. PMID: 27595140
- Lindenbach BD: What's next for hepatitis C virus research? Hepatology. 2016 May; 2016 Mar 19. PMID: 26853717
- Pirakitikulr N, Kohlway A, Lindenbach BD, Pyle AM: The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures. Mol Cell. 2016 Apr 7; 2016 Feb 25. PMID: 26924328
- Binka M, Paintsil E, Patel A, Lindenbach BD, Heimer R: Survival of Hepatitis C Virus in Syringes Is Dependent on the Design of the Syringe-Needle and Dead Space Volume. PLoS One. 2015; 2015 Nov 4. PMID: 26536599
- Kazakov 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 Pathog. 2015 Apr; 2015 Apr 13. PMID: 25875808
- Isken O, Langerwisch U, Jirasko V, Rehders D, Redecke L, Ramanathan H, Lindenbach BD, Bartenschlager R, Tautz N: A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication. PLoS Pathog. 2015 Mar; 2015 Mar 16. PMID: 25774920
- Binka M, Paintsil E, Patel A, Lindenbach BD, Heimer R: Disinfection of syringes contaminated with hepatitis C virus by rinsing with household products. Open Forum Infect Dis. 2015 Jan; 2015 Feb 23. PMID: 26034767
- Kohlway A, Pirakitikulr N, Ding SC, Yang F, Luo D, Lindenbach BD, Pyle AM: The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus. J Virol. 2014 Sep; 2014 Jun 25. PMID: 24965468
- Cordek DG, Croom-Perez TJ, Hwang J, Hargittai MR, Subba-Reddy CV, Han Q, Lodeiro MF, Ning G, McCrory TS, Arnold JJ, Koc H, Lindenbach BD, Showalter SA, Cameron CE: Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein. J Biol Chem. 2014 Aug 29; 2014 Jul 16. PMID: 25031324
- Paintsil E, Binka M, Patel A, Lindenbach BD, Heimer R: 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 15; 2013 Nov 23. PMID: 24273176
- Kohlway 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. J Virol. 2014 Jan; 2013 Oct 30. PMID: 24173222
- Lindenbach BD, Rice CM: The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol. 2013 Oct; 2013 Sep 10. PMID: 24018384
- Lindenbach BD: Virion assembly and release. Curr Top Microbiol Immunol. 2013. PMID: 23463202
- Luo D, Ding SC, Vela A, Kohlway A, Lindenbach BD, Pyle AM: Structural insights into RNA recognition by RIG-I. Cell. 2011 Oct 14. PMID: 22000018
- Counihan NA, Rawlinson SM, Lindenbach BD: Trafficking of hepatitis C virus core protein during virus particle assembly. PLoS Pathog. 2011 Oct; 2011 Oct 20. PMID: 22028650
- Stapleford KA, Lindenbach BD: Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol. 2011 Feb; 2010 Dec 8. PMID: 21147927
- Phan 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. J Virol. 2011 Feb; 2010 Nov 3. PMID: 21047963
- Lindenbach BD: Understanding how hepatitis C virus builds its unctuous home. Cell Host Microbe. 2011 Jan 20. PMID: 21238940
- Paintsil E, He H, Peters C, Lindenbach BD, Heimer R: Survival of hepatitis C virus in syringes: implication for transmission among injection drug users. J Infect Dis. 2010 Oct 1. PMID: 20726768
- Harding MJ, Lepus CM, Gibson TF, Shepherd BR, Gerber SA, Graham M, Paturzo FX, Rahner C, Madri JA, Bothwell AL, Lindenbach BD, Pober JS: An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice. PLoS One. 2010 Apr 1; 2010 Apr 1. PMID: 20376322
- Cheng Y, Tsou LK, Cai J, Aya T, Dutschman GE, Gullen EA, Grill SP, Chen AP, Lindenbach BD, Hamilton AD, Cheng YC: A novel class of meso-tetrakis-porphyrin derivatives exhibits potent activities against hepatitis C virus genotype 1b replicons in vitro. Antimicrob Agents Chemother. 2010 Jan; 2009 Nov 9. PMID: 19901090
- 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. J Virol. 2009 Sep; 2009 Jun 10. PMID: 19515772
- Beran RK, Lindenbach BD, Pyle AM: The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase. J Virol. 2009 Apr; 2009 Jan 19. PMID: 19153239
- Lindenbach BD: Measuring HCV infectivity produced in cell culture and in vivo. Methods Mol Biol. 2009. PMID: 19009272
- Lindenbach BD, Prágai BM, Montserret R, Beran RK, Pyle AM, Penin F, Rice CM: The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J Virol. 2007 Sep; 2007 Jun 20. PMID: 17581983