HIV RGB: Automated Single-Cell Analysis of HIV-1 Rev-Dependent RNA Nuclear Export and Translation Using Image Processing in KNIME

Viruses. 2022 Apr 26;14(5):903. doi: 10.3390/v14050903.


Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus-host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, "Human Immunodeficiency Virus Red-Green-Blue" (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time. Differentially tagged fluorescent viral RNA and protein species are recorded using multicolor long-term (>24 h) time-lapse video microscopy, followed by image processing using a new open-source computational imaging workflow dubbed "Nuclear Ring Segmentation Analysis and Tracking" (NR-SAT) based on ImageJ plugins that have been integrated into the Konstanz Information Miner (KNIME) analytics platform. We describe a typical HIV RGB experimental setup, detail the image acquisition and NR-SAT workflow accompanied by a step-by-step tutorial, and demonstrate a use case wherein we test the effects of perturbing subcellular localization of the Rev protein, which is essential for viral US RNA nuclear export, on the kinetics of HIV-1 late-stage gene regulation. Collectively, HIV RGB represents a powerful platform for single-cell studies of HIV-1 post-transcriptional RNA regulation. Moreover, we discuss how similar NR-SAT-based design principles and open-source tools might be readily adapted to study a broad range of dynamic viral or cellular processes.

Keywords: CRM1; Exportin-1; Gag; ImageJ; KNIME; Konstanz Information Miner; Nuclear Ring Segmentation Analysis and Tracking (NR-SAT); RNA nuclear export; RNA regulation; RNA trafficking; Rev; Rev response element; XPO1; chromosomal region maintenance-1; human immunodeficiency virus type 1; image analysis; live cell imaging; open-source; retrovirus; translation; unspliced RNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • HIV Infections*
  • HIV Seropositivity*
  • HIV-1* / physiology
  • Humans
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Single-Cell Analysis
  • rev Gene Products, Human Immunodeficiency Virus / genetics
  • rev Gene Products, Human Immunodeficiency Virus / metabolism


  • RNA, Viral
  • rev Gene Products, Human Immunodeficiency Virus