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.
Highlights
Most retroviruses share the same characteristics: a spherical capsid about 110–120 nm in diameter enclosing a positive single-strand RNA genome
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
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
Summary
Most retroviruses share the same characteristics: a spherical capsid about 110–120 nm in diameter enclosing a positive single-strand RNA genome. The combination of these two techniques provided a three-dimensional vision of cells revealing, for the first time, the presence of HIV-1 gRNA dimers in the cytosol [15]. These new data support the notion that viral RNA competent for packaging is selected early in the cytoplasm. In order to understand the fundamental mechanisms of retroviral replication, it is essential to accurately characterize the identity and amount of RNA packaged into virions This includes the gRNA, the key molecule carrying all the viral genetic information, and RNA from the host cell, which may contribute to the viral lifecycle [12]. The advantages and limitations will be discussed in terms of performance, accuracy and practical use in the laboratory
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