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Development of a Conditional Replication System Using a Lassa Virus Glycoprotein Complex-Encoding Retroviral Vector
Manya Bakatumana Hans1,2,3, Anita Moendat Fanto1,3, Tsutomu Fukuda4,5
1Department of Clinical Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
Abstract:
A high-risk infectious disease or a Category A pathogen, Lassa virus (LASV), requires strict containment, classified as biosafety level 4 (BSL-4) conditions, which restricts research on the virus due to the scarcity of BSL-4 facilities. Thus, replication-defective pseudotyped retroviral vectors have been widely used as safe materials for neutralizing activity assays of drugs and antibodies in BSL-2. Here, we established a novel retroviral vector system encoding LASV glycoprotein complex (GPC) that can exclusively replicate in cells expressing the Gag-Pol protein of murine leukemia virus (MLV) under BSL-2 conditions. Using this conditional replication system, we successfully isolated LASV GPC variants resistant to either an anti-LASV compound, lamellarin α 20-sulfate, or a neutralizing antibody derived from a Lassa fever survivor. In the lamellarin α 20-sulfate-resistant variants, K125E and H13R amino acid substitutions cooperatively conferred resistance. The K125E enhanced infectivity and simultaneously conferred a lethal effect on cells in the conditional replication system, while the H13R mitigated the latter effect, thereby enabling stable expression of LASV GPC in cells. In the neutralizing antibody-resistant variants, I403T substitution was responsible for the resistance by impairing antibody binding. This study provides a valuable BSL-2-based platform for isolating LASV GPC variants resistant to inhibitors and characterizing their mutations.
Insights
Researchers developed a safe, BSL-2 adaptable retroviral vector for studying Lassa virus (LASV) glycoprotein complex (GPC). This system identified LASV GPC variants resistant to antiviral drugs and antibodies, aiding future therapeutic development.
Area of Science:
- Virology and Infectious Diseases
- Molecular Biology
- Drug Discovery
Background:
- Lassa virus (LASV), a high-risk pathogen, necessitates Biosafety Level 4 (BSL-4) containment, limiting research accessibility due to facility scarcity.
- Replication-defective pseudotyped retroviral vectors are utilized in Biosafety Level 2 (BSL-2) settings for assays, offering a safer alternative for studying LASV.
- Developing accessible research platforms is crucial for advancing understanding and countermeasure development against LASV.
Purpose of the Study:
- To establish a novel, conditionally replicating retroviral vector system for Lassa virus glycoprotein complex (GPC) research under BSL-2 conditions.
- To utilize this system for isolating and characterizing LASV GPC variants resistant to antiviral compounds and neutralizing antibodies.
- To provide a valuable platform for identifying mutations conferring resistance to LASV inhibitors.
Main Methods:
- Development of a novel retroviral vector encoding LASV GPC, designed for conditional replication dependent on murine leukemia virus (MLV) Gag-Pol expression.
- Application of the BSL-2 based conditional replication system to isolate LASV GPC variants resistant to lamellarin α 20-sulfate and a survivor-derived neutralizing antibody.
- Amino acid substitution analysis (K125E, H13R, I403T) to identify mutations responsible for conferring resistance and affecting viral properties.
Main Results:
- Successfully established a BSL-2 compatible retroviral vector system for studying LASV GPC.
- Isolated lamellarin α 20-sulfate-resistant LASV GPC variants with cooperative K125E and H13R mutations, where K125E enhanced infectivity and H13R mitigated lethality.
- Identified the I403T substitution in antibody-resistant variants, which impairs neutralizing antibody binding.
Conclusions:
- The developed conditional replication system provides a safe and effective BSL-2 platform for LASV GPC research.
- This platform facilitates the isolation and characterization of LASV GPC variants resistant to antiviral drugs and antibodies.
- The identified mutations offer insights into LASV GPC-inhibitor interactions and potential resistance mechanisms.
