Researcher, Writer, Academic

Western Blotting was conducted to investigate CANX and CALR protein expression in the context of RSV infection along with siRNA transfection to knockdown expression of CANX and CALR in cells and assess viral replication by Western blot.

Results thus far: RSV infection leads to time-dependent upregulation of ER chaperone proteins CANX and CALR. CALR expression spikes rapidly (~15-fold upregulation at 1 hpi). However, this effect is transient and level return to normal by 24 hpi. CANX expression shows a moderate ~3.5-fold increase at 1 hpi, but this effect is sustained through 24 hpi.

A TALE OF TWO CHAPERONES: CALR exhibits a rapid, transient spike consistent with an acute ER stress response, whereas CANX shows a more moderate but sustained increase. This suggests distinct roles for these two chaperone proteins in the cellular response to RSV infection.

siRNA knockdowns assessed by PCR. Cells were mock treated or transfected with scrambled siRNA, CANX, or CALR siRNA. RNA was isolated 24 hours post transfection and reverse transcribed into cDNA. PCR was carried out using primers sets for GAPDH (control) and CANX/ CALR. Lanes 3,4,5,6 (GAPDH), *lane 5:no band was visible due to experimental error. Lanes 7,8,9,10 (CANX), and lanes 11,12,13,14 (CALR). Potential CANX and CALR knockdown visible in lanes 10 and 14, however loading order is questionable.

Future Directions:

1. Assess the effect of CANX and CALR knockdown on RSV replication to determine whether productive infection is dependent on their upregulation : Confirm siRNA knockdown of these proteins via PCR : Measure RSV protein expression following knockdown via Western blot
2. Determine whether CANX and CALR are regulated at the transcriptional or translational level to determine whether changes in expression are due to a host-driven stress response or direct viral manipulation
3. Perform qPCR on RNA isolated from infection time course to assess changes in mRNA expression