# Development of a Conditional Replication System Using a Lassa Virus Glycoprotein Complex-Encoding Retroviral Vector for Isolating Resistant Variants to Inhibitors in BSL-2

## TL;DR

Scientists created a safe system to study Lassa virus in lower biosafety labs and found mutations that make the virus resistant to treatments.

## Contribution

A novel BSL-2 retroviral vector system for isolating Lassa virus glycoprotein complex variants resistant to inhibitors.

## Key findings

- A retroviral vector encoding Lassa virus glycoprotein complex (GPC) was developed for replication in BSL-2 conditions.
- Lassa virus GPC variants resistant to lamellarin α 20-sulfate and a neutralizing antibody were successfully isolated.
- Specific amino acid substitutions (K125E, H13R, I403T) conferred resistance and altered infectivity or antibody binding.

## 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.