Article ; Online: HIV RGB: Automated Single-Cell Analysis of HIV-1 Rev-Dependent RNA Nuclear Export and Translation Using Image Processing in KNIME.
2022 Volume 14, Issue 5
Abstract: 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. ...
| Abstract | 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. |
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| MeSH term(s) | 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 |
| Chemical Substances | RNA, Viral ; rev Gene Products, Human Immunodeficiency Virus |
| Language | English |
| Publishing date | 2022-04-26 |
| Publishing country | Switzerland |
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't |
| ZDB-ID | 2516098-9 |
| ISSN | 1999-4915 ; 1999-4915 |
| ISSN (online) | 1999-4915 |
| ISSN | 1999-4915 |
| DOI | 10.3390/v14050903 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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