title: From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging.
abstract: In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.
authors.affiliation: CPBS, CNRS, Université de Montpellier, 1919 Route de Mende, Montpellier 34293, France. mireia.dream@gmail.com.
authors.forename: Mireia
authors.initials: M
authors.lastname: Ferrer
authors.affiliation: Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen 5018, Norway. Simon.Henriet@uib.no.
authors.forename: Simon
authors.initials: S
authors.lastname: Henriet
authors.affiliation: CPBS, CNRS, Université de Montpellier, 1919 Route de Mende, Montpellier 34293, France. celia.chamontin@cpbs.cnrs.fr.
authors.forename: Célia
authors.initials: C
authors.lastname: Chamontin
authors.affiliation: CPBS, CNRS, Université de Montpellier, 1919 Route de Mende, Montpellier 34293, France. sebastien.laine@cpbs.cnrs.fr.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lainé
authors.affiliation: CPBS, CNRS, Université de Montpellier, 1919 Route de Mende, Montpellier 34293, France. marylene.mougel@cpbs.cnrs.fr.
authors.forename: Marylène
authors.initials: M
authors.lastname: Mougel
mesh_terms: D000818:Animals; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D012367:RNA, Viral / Q000378:metabolism*; D012190:Retroviridae / Q000502:physiology*; D014771:Virion / Q000378:metabolism*; D014773:Virology / Q000379:methods*; D019065:Virus Assembly
chemical_list: D012367:RNA, Viral
keywords: Gag; HIV-1; Northern blot; RNA; RNA imaging; RT-qPCR; assembly; fluorescence microscopy; packaging; retrovirus