Evaluating the Biological Role of Lassa Viral Z Protein-Mediated RIG-I Inhibition Using a Replication-Competent
Da Di1, Qinfeng Huang1, Hinh Ly1
1Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota, USA.
Abstract:
Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.
Insights
Lassa virus Z protein enhances viral infection by suppressing RIG-I immune response. This study reveals LASV Z as a potential virulence factor and antiviral target.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Lassa virus (LASV) causes lethal hemorrhagic fever with limited treatments, often linked to immune suppression.
- The LASV matrix Z protein inhibits RIG-I-like receptors (RLRs), crucial for innate immunity, but its role in infection was unclear.
- Nonpathogenic Pichinde virus (PICV) Z protein does not inhibit RLRs.
Purpose of the Study:
- To investigate the biological significance of LASV Z-mediated RIG-I inhibition during viral infection.
- To develop a system for controlled RIG-I activation and study its interaction with viral proteins.
- To assess LASV Z as a potential virulence factor in an infectious arenavirus model.
Main Methods:
- Generated a doxycycline-inducible HeLa cell line (HeLa-iRIGN) for controlled RIG-I activation and type I interferon production.
- Created recombinant tri-segmented Pichinde viruses encoding either LASV Z or PICV Z as an additional, nonessential mScarlet fusion protein.
- Analyzed viral gene expression, infection levels, and viral growth in different cell lines (HeLa-iRIGN, Vero, A549) with and without RIG-I induction.
Main Results:
- LASV Z expression, compared to PICV Z, led to higher viral gene expression and increased viral infection in HeLa-iRIGN cells, particularly upon doxycycline induction.
- LASV Z significantly enhanced and prolonged viral gene expression in IFN-competent A549 cells.
- Viral growth curves in Vero and A549 cells were not significantly altered by the heterologous Z gene, suggesting a specific role in immune evasion.
Conclusions:
- LASV Z protein acts as a virulence factor by inhibiting RIG-I-mediated innate immune responses.
- The developed HeLa-iRIGN cell system provides a valuable tool for studying RIG-I signaling and its antagonists.
- LASV Z is a potential target for antiviral therapies against Lassa fever.
