Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

Abstract

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years 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 Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ 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 Ca2+/CaM during viral pathogenesis.

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction

“…Corresponding antibody was used to detect the prey protein in Western blotting, depending on the bait antibody [41]. , and grip strength (C) from 8-week-old wild-type and mdx mice, nonexercised (Non) or after 4 weeks of swimming exercise, 1 h (Ex 1h) or 2 days (Ex 2d) after the last session.…”

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

“…In a separate experiment using the sorted MA104 GCaMP5G/RCEPIA er cells, we were able to observe RCEPIAer localized to ring-like structures (Figure 4I–J), which are morphologically similar to the ER-derived membranes around RV replication complexes (called viroplasms) (30); however, since this was not seen in all infected cells (even at high MOIs) it is also possible this is related to RV-induced disruption in ER-Golgi trafficking (31). Alternatively, and a possible caveat for the use of GECIs in all viral systems, is the recent identification of a CaM binding site on the RV structural protein VP6 (32), which could cause sequestration of RCEPIA er . At the resolution used for these experiments, we were not able to determine whether the diffuse and punctate RCEPIA er displayed different responses to Ca 2+ transients, which would indicate they are distinct pools within the cell.…”

Rotavirus activates a noncanonical ATM‐Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics

“…The time of virus removal was taken as 0 hpi for all experiments (Jolly et al, 2000;Chattopadhyay et al, 2013). At 70% confluency of cells, the growth medium was removed, cells were washed with phosphate-buffered saline (PBS), and incubated with the preactivated RV-SA11 at a multiplicity of infection of 3 (unless otherwise mentioned) for 60 min at 37 C. After adsorption, infectious medium was removed, cells were washed with PBS, supplemented with serum-free medium, and incubated at 37 C in 5% CO 2 .…”