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Published on: January 19, 2024
Identification of cellular calcium binding protein calmodulin as a regulator of rotavirus A infection during
Shiladitya Chattopadhyay1, Trayambak Basak, Mukti Kant Nayak
1Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
Abstract:
Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5 years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca(2+). Ca(2+)-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca(2+) chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca(2+)/CaM during viral pathogenesis.
Insights
Rotavirus infection alters host cell proteins, revealing calmodulin (CaM) interacts with viral protein VP6. This Ca(2+)-dependent interaction enhances rotavirus (RV) replication, offering potential therapeutic targets.
Area of Science:
- Virology
- Proteomics
- Cell Biology
Background:
- Rotavirus (RV) is a leading cause of diarrheal disease and mortality in young children globally.
- Viral infections trigger host cell responses, including differential regulation of cellular proteins.
Purpose of the Study:
- To identify host cell proteins differentially expressed during rotavirus infection.
- To investigate the role of calmodulin (CaM) in rotavirus pathogenesis.
Main Methods:
- Two-dimensional Difference Gel Electrophoresis (2D-DIGE) and MALDI-TOF/TOF mass spectrometry were employed to analyze protein expression in infected HT-29 cells.
- Validation studies were conducted in HT-29 cells and a BALB/c mice model.
- Protein-protein interactions were assessed using biochemical assays.
Main Results:
- Proteomics analysis identified 32 differentially modulated proteins, with 22 being unique, in rotavirus-infected cells.
- Calmodulin (CaM) was identified as a modulated protein that directly interacts with rotavirus protein VP6 in a calcium-dependent manner.
- Inhibition of CaM or calcium chelation reduced viral titers, indicating a positive role for Ca(2+)-CaM in RV replication.
Conclusions:
- Rotavirus infection induces significant changes in host cell protein expression.
- The cellular Ca(2+)/calmodulin complex plays a crucial role in promoting rotavirus pathogenesis and replication.
- Targeting the Ca(2+)-CaM/VP6 interaction presents a potential strategy for controlling rotavirus infections.
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