Analyzing The Interactions Between The Hiv-1 Genomic Rna And Host Proteins Through Proximity Ligation Assay

Abstract

Backgrounds Human Immunodeficiency Virus type-1 (HIV-1) gene expression is highly complex and tightly regulated. Once the host RNA polymerase II recognizes the viral promoter it drives the synthesis of one single messenger RNA molecule, the 9-kb genomic RNA (gRNA), which first undergoes alternative splicing early during replication. Later on, the gRNA in its unspliced form is used as an mRNA for the synthesis of structural proteins and enzymes but also as the packaged genome. The molecular events that allow the interaction between the gRNA and the cellular machineries for nuclear export, translation or packaging have not yet been fully elucidated. The viral protein Rev has been identified as key viral component involved in nuclear export, translation and encapsidation of the unspliced transcript suggesting that Rev is an active component of different HIV-1 gRNA ribonucleoprotein complexes throughout viral replication. Objectives The aim of this study was to analyze the interactions between the HIV-1 genomic RNA and host proteins through proximity ligation assay (PLA). Methods We adapted the PLA, which is usually used to analyze protein/protein interactions, and characterized interactions between the HIV-1 gRNA and host proteins involved in nuclear export and translation initiation. Conclusions Our results validate the PLA as a simple and useful tool to study HIV-1 gRNA/protein interactions within cells. This protocol also serves to determine the subcellular locations where gRNA/protein interactions occur FONDECYT Postdoctoral Grant 3160091 and FONDECYT Grant 1160176