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Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33
Mirko Buttrini1, Flora De Conto1
1Department of Medicine and Surgery, University of Parma, 43126 Parma, Italy.
Background:
The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells.
Methods:
To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme-linked immunosorbent assays. A two-tailed Student's t-test was applied to evaluate differences between groups.
Results:
RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells.
Conclusions:
These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2.
Insights
Ras Homolog Enriched in Brain-like 1 (RhebL1) protein impacts influenza A virus replication differently in various human cells. RhebL1 acts as a restrictive factor against NWS/33 virus in A549 cells but not LLC-MK2 cells.
Area of Science:
- Cell biology
- Virology
- Molecular biology
Background:
- The Mammalian Target of Rapamycin (mTOR) pathway regulates cellular processes and is activated by Ras Homolog Enriched in Brain (Rheb) proteins.
- Rheb proteins, including Rheb1 and Rheb-like-1 (RhebL1), are encoded by specific genes.
- Limited information exists on RhebL1's role in viral infections, prompting this investigation into its function during influenza A virus infection.
Purpose of the Study:
- To investigate the role of RhebL1 during human influenza A/NWS/33 (NWS/33) virus infection.
- To determine RhebL1's involvement in viral replication and host cell response in different cell types.
Main Methods:
- Utilized indirect immunofluorescence (IIF) and 50% tissue culture infectious dose (TCID50) assays to assess NWS/33 virus replication.
- Employed RNA-mediated RNA interference to silence RhebL1 expression.
- Evaluated RhebL1 expression using IIF, Western blotting, and enzyme-linked immunosorbent assays (ELISAs).
Main Results:
- RhebL1 expression was found to be differential across the studied cell models (LLC-MK2 and A549).
- Silencing RhebL1 increased NWS/33 virus infection in A549 cells but not in LLC-MK2 cells.
- Hyperphosphorylated cytokeratin 8, a marker of NWS/33 infection, increased in RhebL1-depleted A549 cells, but not significantly in LLC-MK2 cells.
Conclusions:
- This study provides the first evidence of endogenous RhebL1 protein involvement in viral infection.
- RhebL1 exhibits a host cell-dependent modulatory role in influenza virus infection.
- RhebL1 acts as a restrictive factor against NWS/33 virus replication in A549 cells, with no observed effect in LLC-MK2 cells.
