Abstract
Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation efficiency (Brandt et al., 2007; Blissenbach et al., 2010; Grewe et al., 2012).
Keywords: HIV, Encapsidation, Packaging, RT-PCR, Viral Particles
Materials and Reagents
I. Cell harvesting and ultracentrifugation
II. Cell fractionation and RNA isolation
III. RT-qPCR
IV. Other materials
Equipment
Procedure
Note: The procedure was established for cell numbers obtained from small 25 cm2 cell culture flasks. Initially, 1.5 million adherent cells were seeded. After transfection/infection and subsequent cultivation the cells reached 80-100% confluence at day of cell harvest (three days later).
Figure 1. Schematic diagram of encapsidation assay workflow. Particle-containing supernatants from infected or transfected cells were ultracentrifuged through a 30% sucrose cushion. RNA isolated from concentrated particles and cytoplasm of the infected/transfected cells was analyzed by RT-qPCR. Encapsidation efficiency was determined as the ratio between HIV-1 copy numbers/ml supernatant and HIV-1 copy numbers/μg cytoplasmic RNA. Depending on the experimental design quantification of p24 levels in the concentrated-particle samples is important to normalize particle-associated RNA levels to the amount of p24/Gag particles in the supernatants.
Recipes
Acknowledgments
This protocol was adapted from our papers: Brandt et al. (2007); Blissenbach et al. (2010); and Grewe et al. (2012). This work was funded by a grant from the German Research Foundation (DFG) to Klaus Überla (Ue45/11-1). Bianca Hoffmann is and Bastian Grewe was supported by a fellowship from the DFG graduate school (GRK 1045). Beside Bianca Hoffmann and Bastian Grewe, Inga Ohs, Maik Blissenbach, Sabine Brandt, Bettina Tippler, Thomas Grunwald, Klaus Überla, Rebecca Konietzny, and Klaus Sure were part of the team which established the methods described.
References
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