Abstract
The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5′ end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.
Highlights
Virus particles of HIV-1, the major cause of the AIDS epidemic, and other members of the lentivirus family of retroviruses contain an RNA genome
(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence or absence (ÀRev) of an HIV-1 rev expression plasmid
Effect of Rev on RNA Packaging in Cells Infected with HIV-1- or simian immunodeficiency virus (SIV)-Based Vectors (A) HIV-1-based vectors. 293T cells stably infected with the VH vector were transfected with Hgpsyn and expression plasmids for VSV-G and HIV-1 tat in the presence or absence (ÀRev) of the HIV-1 rev expression plasmid. (B) SIV-based vectors. 293T cells stably infected with VS-Blas were cotransfected with Sgpsyn and VSV-G and tat expression plasmids in the presence or absence of the rev expression plasmid
Summary
Virus particles of HIV-1, the major cause of the AIDS epidemic, and other members of the lentivirus family of retroviruses contain an RNA genome. During particle formation, the unspliced genomic RNA is packaged in preference to spliced viral mRNAs and a more than 1,000-fold excess of cytoplasmic cellular RNAs. Packaging signals have been identified in the 59UTR of lentiviruses (reviewed in [1]). To exclude encapsidation of spliced viral transcripts, packaging signals of retroviruses are generally located or extent 39 to the major splice donor. In HIV-2 and simian immunodeficiency virus (SIV), the major packaging signals are located upstream of the major splice donor [6,7,8]. It is unclear how selective encapsidation of the genomic RNA of HIV-2 and SIV over spliced viral transcripts is accomplished. Long-distance interactions as observed for the HIV-1 59UTR [9] might lead to different conformations of unspliced and spliced HIV-2 and SIV transcripts favouring preferential encapsidation of the unspliced transcript
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