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.

Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3-to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.In contrast to simple retroviruses, HIV-1, a lentivirus, utilizes several trans-acting regulatory proteins which fulfill important functions throughout the different phases of the lentiviral replication cycle (recently reviewed in references 16, 30, and 34). One of those regulators is the 16-kDa HIV-1 Rev protein, which is required for the expression of the structural proteins during the late phase of the HIV-1 replication cycle. This effect of Rev has been attributed to its nuclear export activity. After initial transcription of the integrated proviral genome, "early" transcripts are subjected to the cellular splicing machinery. Alternative splicing events caused by the complex genome organization of HIV-1 lead to expression of Rev from a multiply spliced mRNA. Once Rev is translated in the cytoplasm, it is imported into the nucleus, where it binds to a viral RNA element, the Rev response element (RRE), which is present on singly spliced and unspliced transcripts. Rev seems to circumvent further splicing and leads to an increase of the cytoplasmic levels of unspliced and singly spliced viral transcripts by tethering these transcripts to the Crm1 export pathway (13, 28; reviewed in reference 36). The unspliced and singly spliced transcripts serve as templates for translation, and the unspliced RNA is also encapsidated into assembling virus particles.The magnitude by which Rev enhances lentiviral RNA levels in the cytoplasm has been controversial. Under some experimental conditions, unspliced viral RNA levels in the cytoplasm were detectable only in the presence of Rev, while others observed just a 4-fold enhancement of these cytoplasmic RNA levels by Rev (2,8,9,12,26,36,43,45). Since Rev was found to stimulate protein levels encoded by the Rev-dependent RNAs to a much larger extent than the cytoplasmic levels of these RNAs, Rev also seems to stimulate translation (6, 25, 35; recently reviewed in reference 17). Consistently, Rev was furthermore shown to enhance the association of the Rev-dependent viral RNA with polysomes (9).In trying to dev...

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In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

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