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Abstract Top

Background

During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein.

Findings

Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly.

Conclusions

Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.

Citation: Grewe B, Ehrhardt K, Hoffmann B, Blissenbach M, Brandt S, et al. (2012) The HIV-1 Rev Protein Enhances Encapsidation of Unspliced and Spliced, RRE-Containing Lentiviral Vector RNA. PLoS ONE 7(11): e48688. doi:10.1371/journal.pone.0048688

Editor: Mark Wainberg, McGill University AIDS Centre, Canada

Received: June 22, 2012; Accepted: September 28, 2012; Published: November 1, 2012

Copyright: © 2012 Grewe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by a grant from the DFG (Ue45/11-1). BG was and BH is supported by a fellowship from the DFG graduate school 1045/2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: Bastian.Grewe@rub.de

¤aCurrent address: Institute of Virology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

¤bCurrent address: Serology/Immunology, Medical Laboratory of Bremen, Bremen, Germany

Introduction Top

Encapsidation of the genomic RNA (gRNA) of human immunodeficiency virus type 1 (HIV-1) is mediated by a specific interaction between the viral Gag protein and an RNA structure in the 5′ untranslated region (5′UTR) called encapsidation signal or Psi (Ψ). This association leads to incorporation of gRNA dimers into Gag/GagPol particles. Whereas the core encapsidation signal is composed of 110 nt partially overlapping the gag start codon it is known that sequences up- and downstream of this sequence also influence the encapsidation efficiency. All in all the entire 5′UTR (335 nt) and approximately 300 nt of gag are important for packaging (reviewed in [1]). Complex alternative splicing of the g