Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role...

Area of Science:

• Molecular Biology
• Virology
• Cellular Stress Response

Background:

• Viral infections, including HIV-1, can induce Endoplasmic Reticulum (ER) stress, characterized by the Unfolded Protein Response (UPR).
• The precise mechanisms of UPR modulation by HIV-1 and its functional consequences remain incompletely understood.
• Accurate methodologies are needed to quantify ER stress and UPR activation in HIV-1-infected cells.

Purpose of the Study:

• To establish and validate protocols for measuring ER stress and UPR activation in T-cells during HIV-1 infection.
• To investigate the functional significance of ER stress and UPR modulation on HIV-1 replication and infectivity.
• To provide a comprehensive toolkit for analyzing UPR dynamics in the context of HIV-1 pathogenesis.

Main Methods:

• Quantification of ER stress using Thioflavin T (ThT) staining to detect protein aggregates.
• Measurement of UPR markers (BiP, p-IRE1, PERK, eIF2α, XBP1 splicing, ATF6 cleavage, ATF4, CHOP, GADD34) via immunoblotting and RT-PCR.
• Assessment of HIV-1 replication and infectivity using knockdown experiments, pharmacological agents, Luciferase reporter assays, p24 ELISA, and reporter cell staining.

Main Results:

• ThT fluorescence was found to correlate with established indicators of UPR activation.
• The study provides protocols demonstrating the impact of ER stress and UPR modulation on HIV-1 gene expression, viral replication, and virion infectivity.
• Established correlations between UPR activation levels and viral dynamics.

Conclusions:

• The described methodologies offer a comprehensive approach to studying UPR pathways during HIV-1 infection.
• These methods facilitate a deeper understanding of the intricate interplay between UPR and HIV-1 pathogenesis.
• The findings highlight the functional significance of UPR modulation in controlling HIV-1 replication and infectivity.