The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation

Abstract

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.

Keywords: NP; NSs; SFTSV; autophagosome; replication.

Figure 1

NSsW61Y regulation of viral replication and autophagosome formation. (A,B) HeLa cells were transfected with plasmids encoding NSs and NSsW61Y for 24 h, followed by infection with SFTSV at an MOI of 0.5 for 24 h. Cell lysates were collected using RIPA lysis buffer and analyzed by Western blot using the indicated antibodies. Quantitative results of expression of NP and NSs were normalized by GAPDH using Image J software. (C,D) NSsW61Y-transfected cells exhibited downregulation of LC3 and p62 levels, as well as NP expression levels. Quantitative results of expression level of LC3II, P62, and NSs were normalized by GAPDH using Image J software. Data from independent experiments are presented as the mean intensity of the protein band ±SEM.

Figure 2

Dose-dependent downregulation of autophagosome marker levels by NSW61Y expression. (A) HeLa cells were overexpressed by transfection with each recombinant plasmid, and the levels of autophagosome-related genes were analyzed by Western blot assay. (C) Plasmids containing each gene were transfected into HeLa cells in a dose-dependent manner for 24 h. After cell lysis, the target protein expression was examined by Western blot. (B,D) Using Image J software, the quantitative expression levels of P62 and LC3II were normalized by GAPDH. The mean band density ±SEM is utilized for displaying results from independent investigations.

Figure 3

Effect of NSW61Y on autophagy-related to the lysosome-dependent degradation pathway. (A,B) Transfected HeLa cells were infected with SFTSV at an MOI of 0.5 for 24 h. Following infection, cells were treated with CQ at 100μM for 6 h, and cell lysates were examined by Western blot and analyzed the expressed protein levels of LC3II/GAPDH and P62/GAPDH. (C,D) Cells transfected for 24 h were incubated with LysoTracker deep red for 1 h before being processed for fluorescence microscopy. Quantitative analysis of fluorescence levels are illustrated and the independent experiments are presented as a mean fluorescence intensity ± SEM. (E,F) HeLa cells were co-transfected with recombinant plasmids of NP, NSs, NSsW61Y, NP-NSs, and NP-NSsW61Y, respectively. Cell lysates were evaluated through a Western blot. Representative quantitation of p-mTOR level was normalized by GAPDH and data from independent experiments are presented as a mean band intensity ±SEM.