Abstract
Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus–host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.
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References
Buchmeier MJ, Peter CJ, de la Torre JC (2007) Arenaviridae: the viruses and their replication, vol 2. Lippincott William and Wilkins, Philadelphia
Enria DA, Briggiler AM, Sanchez Z (2008) Treatment of Argentine hemorrhagic fever. Antivir Res 78:132–139
Barton LL, Mets MB, Beauchamp CL (2002) Lymphocytic choriomeningitis virus: emerging fetal teratogen. Am J Obstet Gynecol 187:1715–1716
Fischer SA, Graham MB, Kuehnert MJ, Kotton CN, Srinivasan A, Marty FM, Comer JA, Guarner J, Paddock CD, DeMeo DL, Shieh WJ, Erickson BR, Bandy U, DeMaria A Jr, Davis JP, Delmonico FL, Pavlin B, Likos A, Vincent MJ, Sealy TK, Goldsmith CS, Jernigan DB, Rollin PE, Packard MM, Patel M, Rowland C, Helfand RF, Nichol ST, Fishman JA, Ksiazek T, Zaki SR (2006) Transmission of lymphocytic choriomeningitis virus by organ transplantation. N Engl J Med 354:2235–2249
Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, Jahrling PB, Ksiazek T, Johnson KM, Meyerhoff A, O’Toole T, Ascher MS, Bartlett J, Breman JG, Eitzen EM Jr, Hamburg M, Hauer J, Henderson DA, Johnson RT, Kwik G, Layton M, Lillibridge S, Nabel GJ, Osterholm MT, Perl TM, Russell P, Tonat K (2002) Hemorrhagic fever viruses as biological weapons: medical and public health management. JAMA 287:2391–2405
Birmingham K, Kenyon G (2001) Lassa fever is unheralded problem in West Africa. Nat Med 7:878
Gunther S, Lenz O (2004) Lassa virus. Crit Rev Clin Lab Sci 41:339–390
Freedman DO, Woodall J (1999) Emerging infectious diseases and risk to the traveler. Med Clin North Am 83:865–883
Richmond JK, Baglole DJ (2003) Lassa fever: epidemiology, clinical features, and social consequences. BMJ 327:1271–1275
Briese T, Paweska JT, McMullan LK, Hutchison SK, Street C, Palacios G, Khristova ML, Weyer J, Swanepoel R, Egholm M, Nichol ST, Lipkin WI (2009) Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS Pathog 5:e1000455
Damonte EB, Coto CE (2002) Treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy. Adv Virus Res 58:125–155
Lee KJ, Novella IS, Teng MN, Oldstone MB, de La Torre JC (2000) NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs. J Virol 74:3470–3477
Perez M, Craven RC, de la Torre JC (2003) The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci U S A 100:12978–12983
Ortiz-Riano E, Cheng BY, de la Torre JC, Martinez-Sobrido L (2012) Self-association of lymphocytic choriomeningitis virus nucleoprotein is mediated by its N-terminal region and is not required for its anti-interferon function. J Virol 86:3307–3317
Ortiz-Riano E, Cheng BY, de la Torre JC, Martinez-Sobrido L (2011) The C-terminal region of lymphocytic choriomeningitis virus nucleoprotein contains distinct and segregable functional domains involved in NP-Z interaction and counteraction of the type I interferon response. J Virol 85:13038–13048
Pythoud C, Rodrigo WW, Pasqual G, Rothenberger S, Martinez-Sobrido L, de la Torre JC, Kunz S (2012) Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKepsilon. J Virol 86:7728–7738
Martinez-Sobrido L, Emonet S, Giannakas P, Cubitt B, Garcia-Sastre A, de la Torre JC (2009) Identification of amino acid residues critical for the anti-interferon activity of the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus. J Virol 83:11330–11340
Martinez-Sobrido L, Giannakas P, Cubitt B, Garcia-Sastre A, de la Torre JC (2007) Differential inhibition of type I interferon induction by arenavirus nucleoproteins. J Virol 81:12696–12703
Martinez-Sobrido L, Zuniga EI, Rosario D, Garcia-Sastre A, de la Torre JC (2006) Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus. J Virol 80:9192–9199
Borrow P, Martinez-Sobrido L, de la Torre JC (2010) Inhibition of the type I interferon antiviral response during arenavirus infection. Viruses 2:2443–2480
Pythoud C, Rothenberger S, Martinez-Sobrido L, de la Torre JC, Kunz S (2015) Lymphocytic Choriomeningitis virus differentially affects the virus-induced type I interferon response and mitochondrial apoptosis mediated by RIG-I/MAVS. J Virol 89:6240–6250
Rodrigo WW, Ortiz-Riano E, Pythoud C, Kunz S, de la Torre JC, Martinez-Sobrido L (2012) Arenavirus nucleoproteins prevent activation of nuclear factor kappa B. J Virol 86:8185–8197
Kunz S, Borrow P, Oldstone MB (2002) Receptor structure, binding, and cell entry of arenaviruses. Curr Top Microbiol Immunol 262:111–137
Radoshitzky SR, Abraham J, Spiropoulou CF, Kuhn JH, Nguyen D, Li W, Nagel J, Schmidt PJ, Nunberg JH, Andrews NC, Farzan M, Choe H (2007) Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses. Nature 446:92–96
Capul AA, Perez M, Burke E, Kunz S, Buchmeier MJ, de la Torre JC (2007) Arenavirus Z-glycoprotein association requires Z myristoylation but not functional RING or late domains. J Virol 81:9451–9460
Cornu TI, de la Torre JC (2001) RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome. J Virol 75:9415–9426
Capul AA, de la Torre JC (2008) A cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding. Virology 382:107–114
Perez M, Greenwald DL, de la Torre JC (2004) Myristoylation of the RING finger Z protein is essential for arenavirus budding. J Virol 78:11443–11448
Emonet SE, Urata S, de la Torre JC (2011) Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies. Virology 411:416–425
de la Torre JC (2008) Reverse genetics approaches to combat pathogenic arenaviruses. Antivir Res 80:239–250
Ortiz-Riano E, Ngo N, Devito S, Eggink D, Munger J, Shaw ML, de la Torre JC, Martinez-Sobrido L (2014) Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor. J Virol 88:878–889
Cubitt B, Ortiz-Riano E, Cheng BY, Kim YJ, Yeh CD, Chen CZ, Southall NOE, Zheng W, Martinez-Sobrido L, de la Torre JC (2020) A cell-based, infectious-free, platform to identify inhibitors of Lassa virus ribonucleoprotein (vRNP) activity. Antivir Res 173:104667
Ortiz-Riano E, Cheng BY, de la Torre JC, Martinez-Sobrido L (2012) D471G mutation in LCMV-NP affects its ability to self-associate and results in a dominant negative effect in viral RNA synthesis. Viruses 4:2137–2161
Ortiz-Riano E, Cheng BY, Carlos de la Torre J, Martinez-Sobrido L (2013) Arenavirus reverse genetics for vaccine development. J Gen Virol 94:1175–1188
Cheng BY, Ortiz-Riano E, de la Torre JC, Martinez-Sobrido L (2013) Generation of recombinant arenavirus for vaccine development in FDA-approved Vero cells. J Vis Exp. https://doi.org/10.3791/50662
Cheng BY, Ortiz-Riano E, Nogales A, de la Torre JC, Martinez-Sobrido L (2015) Development of live-attenuated arenavirus vaccines based on codon deoptimization. J Virol. https://doi.org/10.1128/JVI.03401-14
Rodrigo WW, de la Torre JC, Martinez-Sobrido L (2011) Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses. J Virol 85:1684–1695
Emonet SF, Garidou L, McGavern DB, de la Torre JC (2009) Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest. Proc Natl Acad Sci U S A 106:3473–3478
Cheng BY, Ortiz-Riano E, de la Torre JC, Martinez-Sobrido L (2015) Arenavirus genome rearrangement for the development of live-attenuated vaccines. J Virol. https://doi.org/10.1128/JVI.00307-15
Ye C, de la Torre JC, Martinez-Sobrido L (2020) Development of reverse genetics for the prototype New World mammarenavirus Tacaribe virus. J Virol 94
Cai Y, Iwasaki M, Beitzel BF, Yu S, Postnikova EN, Cubitt B, DeWald LE, Radoshitzky SR, Bollinger L, Jahrling PB, Palacios GF, de la Torre JC, Kuhn JH (2018) Recombinant Lassa virus expressing green fluorescent protein as a tool for high-throughput drug screens and neutralizing antibody assays. Viruses 10
Flatz L, Hegazy AN, Bergthaler A, Verschoor A, Claus C, Fernandez M, Gattinoni L, Johnson S, Kreppel F, Kochanek S, Broek M, Radbruch A, Levy F, Lambert PH, Siegrist CA, Restifo NP, Lohning M, Ochsenbein AF, Nabel GJ, Pinschewer DD (2010) Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity. Nat Med 16:339–345
Yun NE, Seregin AV, Walker DH, Popov VL, Walker AG, Smith JN, Miller M, de la Torre JC, Smith JK, Borisevich V, Fair JN, Wauquier N, Grant DS, Bockarie B, Bente D, Paessler S (2013) Mice lacking functional STAT1 are highly susceptible to lethal infection with Lassa virus. J Virol 87:10908–10911
Albarino CG, Bird BH, Chakrabarti AK, Dodd KA, Erickson BR, Nichol ST (2011) Efficient rescue of recombinant Lassa virus reveals the influence of S segment noncoding regions on virus replication and virulence. J Virol 85:4020–4024
Acknowledgments
We thank past and present members in L.M-S and J.C.T laboratories of the development of mammarenavirus reverse genetics techniques and plasmids, including LASV. Mammarenavirus research in L.M-S laboratory was funded by the National Institutes of Health (NIH) R03AI099681, R21AI119775, R21AI128097-01, R21AI121550, R21AI1135284, RO1AI142985, and R43AI119775-01 grants and by the Department of Defense (DoD) W81XWH1810071 and W81XWH1910496 grants. Research in J.C.T. laboratory is supported by grants RO1AI047140, RO1AI077719, RO1AI079665, and RO1AI142985.
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Martínez-Sobrido, L., Ye, C., de la Torre, J.C. (2024). Plasmid-Based Lassa Virus Reverse Genetics. In: Pérez, D.R. (eds) Reverse Genetics of RNA Viruses. Methods in Molecular Biology, vol 2733. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3533-9_8
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DOI: https://doi.org/10.1007/978-1-0716-3533-9_8
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