Retroviral RNA elements integrate components of post-transcriptional gene expression

Abstract

Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency, and may even lack pathogenicity, but all retroviruses require cytoplasmic expression of intron-containing mRNA. In the cytoplasm, the primary viral transcript has two essential roles as mRNA template for protein synthesis and as genomic RNA for packaging into progeny virions. Cellular proteins are used by the virus to modulate synthesis, processing, and translation of the viral RNA. To subvert the normal RNA processing cascade and achieve nuclear export of intron-containing viral RNA, retroviruses utilize structured RNA elements and viral or cellular protein partners. These nuclear interactions determine the cytoplasmic fate of viral RNAs by facilitating RNA stability, nuclear export, translational efficiency, and even assembly of progeny virions. The HIV Rev responsive element (RRE) and Rev protein have been a informative paradigm for dissection of the process of eukaryotic RNA nuclear export. Rev is an adapter protein that bridges RRE-containing RNA and the CRM1 nuclear export receptor, which delivers intron-containing RNA to a nuclear export pathway typically used for 5s rRNA and protein transport. This review summarizes data indicating that Rev/RRE also targets cytoplasmic transcripts to the cytoskeletal polysomes and activates their translational efficiency. The interesting parallel is discussed that genetically simpler retroviruses lack a Rev-like protein and recruit cellular proteins to distinct RNA elements that modulate post-transcriptional gene expression through different export pathways. These pathways include the global mRNA export pathway mediated by Tap, and Tap- and CRM1-independent pathways. The CRM1-independent nuclear export pathway accessed by the spleen necrosis virus post-transcriptional control element is functionally linked to RU5-mediated translational enhancement in the cytoplasm. The simple retroviral post-transcriptional control elements also modulate RNA splicing efficiency, stability, assembly of virions, and subsequent viral egress from the cell. Thus, multiple layers of post-transcriptional control are executed by these retroviral RNA elements, which serve as a compact platform for interaction with nuclear and possibly cytoplasmic protein partners. Further characterization of the cellular partners and their regulation will be an important step to full understanding of nuclear-cytoplasmic connections that hardwire post-transcriptional gene expression in eukaryotic cells.

Introduction

Cytoplasmic expression of intron-containing RNA is a mandatory step in replication of all retroviruses. The unspliced version of the primary RNA transcript serves two essential roles in the retroviral lifecycle as mRNA template for synthesis of viral structural and enzymatic proteins, and as genomic RNA that is packaged into viral particles. Two post-transcriptional obstacles must be overcome to produce progeny virions: nucleo-cytoplasmic export of the intron-containing RNA, and competition between the cellular translation machinery and viral assembly components for the cytoplasmic transcript. Recently, significant progress has been made in understanding structured RNA motifs that retroviruses utilize to recruit cellular proteins and achieve nuclear export and cytoplasmic expression of intron-containing RNA. This article will begin by contrasting the post-transcriptional processing cascade of typical cellular mRNAs and intron-containing retroviral RNAs, with emphasis on linkage between steps in the cascade. Secondly, the article briefly overviews RRE/Rev transactivation of nuclear export and summarizes experiments that support activation by Rev of the translational efficiency of RRE-containing transcripts. Lastly, recent studies are summarized of three other retroviral RNA elements from genetically simpler retroviruses that subvert the cascade and mediate cytoplasmic expression of intron-containing viral transcripts.

Nascent RNAs are co-transcriptionally programmed for RNA processing

Early in the process of transcription, protein-protein and RNA-protein interactions program pre-mRNAs for processing into mature mRNAs [1;2]. During formation of the transcription initiation complex at the core promoter, RNA processing factors are recruited to the C-terminal domain (CTD) of RNA polymerase II. The transition from the initiation phase to the elongation phase of transcription is triggered by phosphorylation of the CTD and is marked by release of initiation factors and recruitment of additional processing factors. RNA processing begins as the CTD delivers guanine transferase and methylase to the 5′ RNA terminus just as the first 25 nucleotides of nascent RNA emerge from the transcription complex. Addition of a 7-methyl guanine cap and subsequent intron removal occur during transcription elongation. Like capping enzymes, splicing factors may be delivered to the nascent RNA by the CTD. Studies with the fibronectin gene have revealed promoter-specific variation in intron definition that produces alternatively spliced transcripts [3]. The production of the alternatively spliced RNAs has been attributed to differences in the constellation of splicing regulatory proteins recruited to the CTD by the various core promoters [4]. Typical cellular RNAs require intron removal for efficient nuclear export and evidence is mounting that nuclear interactions with RNA splicing factors license the RNA for export to the cytoplasm [5-9].

Retroviruses, however, subvert this aspect of the cellular RNA processing cascade and achieve cytoplasmic expression of both spliced and intron-containing viral transcripts. Retroviruses modulate intron definition to achieve balanced expression of spliced and intron-containing RNAs, both of which are needed to produce progeny virions [10;11]. Balanced expression is coordinated by RNA binding proteins that interact with a combination of retroviral exonic splicing enhancers, silencers, and suboptimal splice sites [12-17]. Because splicing occurs cotranscriptionally, the retroviral commitment to produce stable intron-containing RNA is expected to occur during transcription.

The final step in maturation of pre-mRNA, 3′ end processing, is modulated by a 3′ terminal AAUAAA consensus sequence. Polyadenylation is orchestrated by cellular factors that are delivered to the RNA in association with the CTD and the 5′ RNA cap [18]. The mature mRNA is exported though the nuclear pore complex to the cytoplasm by shuttling RNA binding proteins, nucleoporins and other cellular proteins [19]. Particular RNA export pathways are utilized for particular classes of cellular RNAs [20] and are a conduit for flow of quality control signals between the nucleus and cytoplasm. In addition to nuclear export licensing, nuclear factors assembled onto the spliced RNA program the RNA for cytoplasmic translation [21;22] and possibly nonsense mediated decay [23;24].

RNA elements in retroviruses choreograph nucleocytoplasmic communication that directs efficient cytoplasmic expression of intron-containing retroviral mRNAs. The HIV Rev responsive element (RRE) and its cognate viral shuttle protein Rev have been a cornerstone model system for illumination of the process of eukaryotic nuclear export.

The following sections briefly summarize the role of RRE/Rev in nuclear export and then review data that support Rev activation of the translational efficiency of RRE-containing RNA. Comparison is made between RRE/Rev and RNA elements in genetically simpler retroviruses that recruit cellular proteins that, like virally encoded Rev, modulate nuclear export and efficient cytoplasmic expression of intron-containing RNA. Recent findings with these RNA elements highlight the notion that RNA synthesis, processing, nuclear export and cytoplasmic translation are functionally linked steps in a cascade that is necessary for accurate gene expression. Understanding the differences between the elements and their protein partners will illuminate cellular mechanisms that choreograph post-transcriptional gene expression.

Nuclear interaction between RRE and Rev activates cytoplasmic expression of intron-containing RNA

The RRE is an elaborate stem-loop structure present within intronic sequences of HIV transcripts that coordinates interaction with Rev, a 116 amino acid nuclear-cytoplasmic shuttling RNA binding protein (reviewed in [19;25;26]). Rev contains an arginine-rich nuclear localization signal that facilitates shuttling into the nucleus and subsequent binding to the RRE. The leucine-rich activation domain can function as an autonomous nuclear export signal and is the binding site for the CRM1/exportin 1 nuclear export receptor. Multimerization of Rev monomers at the RRE is necessary and Rev acts as an adapter protein that recruits a collection of CRM1 molecules to the complex. CRM1 mediates delivery of the RRE-Rev complex to the nuclear pore and nuclear export to the cytoplasm by a pathway normally used for export of 5s rRNA and proteins with leucine-rich nuclear export signals [19] (Figure 1). A co-transcriptional mechanism for Rev transactivation is suggested by the demonstration that RRE/Rev activation is specific for newly synthesized RNAs and is dependent on the transcription rate of the RNA [27]. RRE/Rev may choreograph cotranscriptional interactions that contribute to licensing the mRNA for nuclear export.

Fig. 1.

Summary of retroviral post-transcriptional RNA elements and their different roles. Four distinct retroviral structured RNA elements have been identified that interact in the nucleus with selected viral and cellular shuttling RNA binding proteins and direct cytoplasmic expression of intron-containing RNA. HIV Rev responsive element (RRE)-containing RNAs interact with viral Rev protein that is an adapter protein to nuclear export receptor CRM1 and activates export by a pathway generally used for export of 5s rRNA and protein with leucine-rich nuclear export signal. RRE/Rev also facilitate RNA stability and translation. RNAs that contain the simian retrovirus type 1 or Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE), as well as cellular polyadenylated mRNAs, achieve nuclear export by interaction with TAP and cofactor p15/NXT1. RNA helicase A (RHA) has also been shown to interact with MPMV CTE. The Rous sarcoma virus (RSV) direct repeat (dr) acts as a CTE, but provides access to a distinct nuclear export pathway that is independent of both CRM1 and TAP. The dr also facilitates RNA stability and assembly of viral capsids. RNAs that contain the spleen necrosis virus (SNV) RU5 element are exported independently of CRM1 and exhibits translational enhancement in the cytoplasm.

Extensive experimentation with reporter plasmids and with HIV proviruses in a wide variety of cell types has characterized RRE/Rev as a potent molecular switch that significantly trans-activates cytoplasmic accumulation of these intron-containing RNAs and synthesis of their protein products. In the absence of Rev, cis-acting inhibitory sequences cause nuclear retention and low steady state accumulation of these RNAs [28-32]. In the presence of Rev, the stability and nuclear export of RRE-containing transcripts is activated [33].

Quantitative analysis of RNA levels and protein production has revealed a discordant relationship between the increase in cytoplasmic RNA level and protein production, implying that Rev transactivation also enhances translational efficiency [34;35]. Three experimental approaches have illuminated the effect of Rev on translational efficiency of RRE-containing RNAs. These are quantitative comparisons of cytoplasmic RNA and protein levels in the context of Rev-dependent reporter transcripts; analysis of the polyribosomal accumulation in response to Rev of HIV proviral and reporter transcripts; and subcellular localization of reporter RNAs by confocal microscopy.

Cytoplasmic role for RRE/Rev in translational activation

Quantitative RNA analysis and Gag Western immunoblots were used to compare the effect of Rev on RRE/Rev-dependent gag RNA and Gag protein levels in transfected Hela cells (Hltat) [35]. In response to Rev, cytoplasmic gag RNA levels increased 4- to 16-fold. The increase in Gag protein production was 850-fold, which suggested that increased cytoplasmic accumulation of gag RNA was insufficient to account for the dramatic increase in Gag protein production. Ribosomal profile analysis and RT-PCR was performed to evaluate the quantity of gag RNA associated with the translational machinery in the presence and absence of Rev. The data demonstrated that Rev increased polyribosomal association of the cytoplasmic RNAs. In the absence of Rev, 4% of gag mRNA was associated with the polysomes, whereas in the presence of Rev, 20% was associated with polysomes. Parallel in situ hybridization analysis revealed the RRE-containing transcripts localized to distinct cytoplasmic compartments in response to Rev, which were speculated to be important for the increased translational efficiency of the intron-containing RNA. A caveat of these experiments is that correlation between steady state RNA and protein levels does not take into account differential stability of RNA versus protein. However, these data combined with identification of a distinct shift in subcellular compartmentalization in the in situ analysis support the notion that RRE/Rev activates cytoplasmic accumulation of the Rev-dependent RNAs and facilitates their association with the translational machinery.

A role for RRE/Rev in facilitating translational efficiency was also observed in experiments with nonviral intron-containing reporter RNA that encodes chloramphenicol acetyl transferase (cat) [36]. The cat RNA lacks the nuclear retention sequences found in gag RNA and cat RNA exhibits constitutive export from the nucleus to the cytoplasm. Cytoplasmic expression of intron-containing RRE-containing cat transcripts was evaluated in COS cells in the presence or absence of Rev. Cytoplasmic cat RNA was observed in the absence of Rev and the level remained similar in the presence of Rev. Despite similar nuclear and cytoplasmic cat RNA levels, CAT protein levels increased 3.5-fold in response to Rev consistent with increased translational efficiency of the cat RNA in response to Rev. The magnitude increase in CAT protein production is less than that observed in the context of the retroviral Gag reporter gene. The smaller increase is attributable to lack of nuclear retention sequences and constitutive nuclear export of the cat RNA combined with the higher threshold of CAT protein production in the absence of Rev.

The effect of Rev on proviral HIV transcripts was evaluated in lymphoid cells transiently transfected with high levels of proviral DNAs (500 μg per 10⁸ 729 B cells) that lack or contain the Rev open reading frame [34;37]. RT-PCR data indicated that Rev increased the cytoplasmic accumulation of the unspliced HIV gag RNA by 10-fold. A discordant >450-fold increase in Gag protein production was observed. In the case of singly spliced env/vpu transcripts, cytoplasmic levels were only moderately increased by Rev, but protein production was undetectable unless Rev was present. Polyribosomal profile and RT-PCR analysis revealed that the presence of Rev correlated with a shift in the subcellular localization of these intron-containing gag, vif, vpr, or env/vpu RNA to polyribosomes, indicating that Rev activates the translational efficiency of RRE-containing RNAs.

Contrasting results were obtained in lymphoid cells that were transduced with retroviral vectors that lack or contain a functional rev gene and then selected for integrated single-copy proviruses [33]. S1 nuclease protection assays were used to evaluate the RNA expression levels and revealed that mutation of the Rev open reading frame correlated with less than minimally detectable Rev-dependent transcripts in the cytoplasm and that RRE/Rev is absolutely necessary for cytoplasmic accumulation in transduced lymphoid cells.

A prominent difference between the transient transfection approach by Arrigo et al [34;37], which did not observe a strict requirement for Rev in cytoplasmic accumulation, and the transduction approach by Malim and Cullen [33], which did observe a strict requirement, is the introduction of either multiple copies of episomal HIV DNA as compared to a single provirus. Also the experiments were performed in two different types of lymphoid cells, B or T cells. Overexpression from episomal HIV DNAs may alter the relationship between the HIV transcription products and cellular interactive proteins that modulate nuclear retention of RRE-containing mRNAs. Malim and Cullen (1993) validated this potential role for cellular nuclear retention proteins by experiments in COS cells. Upon vector transduction into COS cells, low but detectable levels of the intron-containing HIV transcripts were observed in the cytoplasm in the absence of Rev, in agreement with Lawrence et al [36]. This difference from the transduced lymphoid cells is attributable to COS-specific factors that derepress nuclear retention and abrogate the block to stable cytoplasmic accumulation of Rev-dependent RNAs. These results highlight the role of cellular co-factors in the Rev-dependence of RRE-containing RNAs and that cell-type specific differences are observed in nuclear retention of these transcripts.

RRE/Rev facilitates cytoplasmic localization to cytoskeletal polysomes

Fluorescence in situ hybridization assays and biochemical analysis have been applied to directly evaluate the hypothetical role of Rev in directing HIV RNA to a translational compartment [38;39]. Overexpression of a rev-defective RRE-containing HIV expression plasmid in Hela cells was used to achieve cytoplasmic accumulation of gag mRNA and validated the notion that high level expression can overcome nuclear retention. In the presence of Rev, cytoplasmic gag RNA increased 5-fold and Gag protein increased up to 200-fold. These results are consistent with the previous conclusions that Rev activates the cytoplasmic accumulation of gag mRNA and also enhances translational efficiency. Rev also caused a change in the subcellular localization of gag mRNA. In the absence of Rev, the RNA formed a diffuse pattern throughout the cytoplasm and nucleus, but was excluded from the nucleolus. In the presence of Rev, nuclear staining was abrogated and clusters of RNA formed in the cytoplasm. Gag protein synthesis was limited to clusters at perinuclear sites and colocalized with an element of the cytoskeleton containing β-actin. The cytoskeleton constitutes a framework for supporting interaction of polyribosomes and mRNA [40;41]. These in situ hybridization results and the previous polyribosome analyses support a model in which Rev facilitates clustering of gag mRNA to cytoskeletal polyribosomes.

In summary, extensive analysis has characterized RRE/Rev as a potent post-transcriptional activator that integrates nuclear export, stability, and translational efficiency of HIV proviral and reporter gene RNAs. RRE/Rev has a necessary and sufficient role in promoting cytoplasmic accumulation of intron-containing HIV RNA. The combined effect by RRE/Rev on nuclear export, stability and translational efficiency dramatically activates protein production.

Recruitment of cellular post-transcriptional factors by RNA elements of simple retroviruses

Genetically simpler retroviruses lack an analogous viral Rev protein to regulate the cytoplasmic expression of their intron-containing RNA. However, RNA elements have been identified in selected simple retroviruses that recruit cellular factors and modulate the stability, nuclear export, translational efficiency and packaging of the unspliced RNA and assembly of viral proteins.

A redundant stem-loop structure comprising 160 nt in the 3′ UTR of Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1) acts as a constitutive transport element (CTE) for intron-containing viral transcripts in Xenopus oocytes and various cell lines [42-46]. CTE function is abrogated in quail cells unless human TAP (hTAP) nuclear export protein is added exogeneously [47] because quail Tap lacks productive interaction at the CTE binding site [48]. CTE interacts directly with hTAP, which in turn, delivers the CTE-containing transcript to the nuclear pore for export to the cytoplasm (Figure 1) [42]. RNA helicase A has also been shown to interact with CTE-containing RNA and may play a role in the nuclear export of the RNA [49].

In Rous sarcoma virus (RSV) and avian sarcoma virus, a structured direct repeat (dr) element, also near the 3′ UTR, integrates nuclear export with additional post-transcriptional functions (Figure 1). Like CTE, dr is a redundant stem-loop structure and is approximately 160 nt in length [50]. Mutational analysis reveals that dr is necessary for stability and cytoplasmic accumulation of intron-containing RNA [50;51;52]. Selected dr mutants exhibit defective viral particle assembly that is manifested by reduced virus release [51;54] or defective RNA packaging function [55;56]. A possible explanation is that dr mutation disrupts interaction with cellular factors that deliver the RNA to a permissive cytoplasmic microenvironment for particle assembly and egress from the cells. Cytoplasmic accumulation of dr-containing transcripts occurs independently of Tap protein and CRM1 [57] and is modulated by a yet to be identified cellular protein. dr does not function as a transport element in the Xenopus ooctye nuclear export assay [57]. Cellular nuclear-cytoplasmic shuttle proteins are the candidate factors that couple the nuclear export and cytoplasmic utilization of the dr-containing RNA. One possible scenario is that dr mediates increased steady state levels of the intron-containing transcripts that squelches interaction with nuclear retention proteins. This is projected to derepress interaction with cellular nuclear-cytoplasmic shuttle proteins that carry the dr-containing RNA cargo through a permissive protein export pathway.

5′ LTR sequences of SNV facilitate cytoplasmic expression of intron-containing RNA

The 5′ long terminal repeat (LTR) of avian spleen necrosis virus (SNV), which is more closely related to murine leukemia viruses than to the avian sarcoma viruses, facilitates RRE/Rev-independent expression of HIV gag reporter RNA in human, dog and monkey cells [58]. The LTR consists of three regions (U3, R, U5) and the 165 nt RU5 region corresponds to the 5′ terminus of the viral RNA. RU5 functions in a position-dependent and orientation-dependent manner and is necessary and sufficient for RRE/Rev-independent gag expression. ELISA results of gag reporter plasmids containing the CMV IE or SNV LTR showed that Gag production is dramatically shifted from undetectable (≤ 0.1 ng) up to 80 ng/ml in response to the SNV sequences. Quantitative RNA analysis by RNase protection assays (RPAs) detect low levels of steady state RNA in the cytoplasm that increase 2 to 4-fold in response to the SNV LTR. RU5 is positioned upstream of the 5′ splice site and both spliced and intron-containing gag reporter transcripts are increased. The discordance between gag RNA accumulation and Gag production implied a possible translational component in response to the SNV LTR. RPAs were used to quantify cytoplasmic gag transcripts that cosedimented with active translational machinery in sucrose gradients and revealed that the RU5-containing gag RNA was 3-fold more abundant in polysomal RNA. Combined effects of RU5 on cytoplasmic accumulation and localization to the translational machinery are sufficient to account for the dramatic increase in Gag production (Figure 1).

The polysome sedimentation assay was also used to compare the effect of RRE/Rev and CTE on polysome association in the context of SV40-based gag reporter plasmids in transfected 293 cells. In the absence of Rev, low but detectable levels of gagRRE RNA were associated with polysomes and the level increased 3-fold in response to Rev. The 3-fold RNA increase corresponded with a shift in Gag production from undetectable (≤ 0.1 ng) to 350 ng/ml. A similar quantity of gagCTE RNA was localized to the translational machinery, but Gag production was only 3 ng/ml. The results indicate that CTE increases cytoplasmic association with polysomes in 293 cells, but the translational efficiency of the RNA is suboptimal. The observation of vastly different Gag levels despite similar polysomal accumulation of gag RNA implies a role for rate-limiting positive-acting translation factor(s) or negative-acting RNA masking factor(s). While not necessary for ribosome association, the putative factors may be necessary for ribosomes to productively transverse the viral RNA template, which contains a series of highly structured replication motifs that include the RNA packaging signal. Such motifs act as structural barriers to ribosome scanning and impede protein synthesis [59-61]. We speculate that RRE/Rev and SNV RU5 recruit an RNA helicase that melts structural barriers and augments translational efficiency. In 293 cells, CTE lacks productive interaction with the putative helicase and protein synthesis remains inefficient.

RU5 is a distinct enhancer of translational initiation

The translational mechanism of SNV RU5 was evaluated further in the context of nonviral luciferase (luc) reporter RNA in 293 cells [62]. The presence of SNV RU5 augmented Luc production 10-fold. RU5 had no effect on cytoplasmic accumulation of luc RNA, which efficiently accumulated in the cytoplasm in the presence or absence of RU5. Ribosome profile and Northern blot analysis revealed that RU5 increased the proportion of luc RNA associated with heavy polysomes from 15% to 50%. The data indicated that RU5 enhances translation initiation 3.5-fold, which is sufficient to account for the 10-fold increase in Luc production. Experiments with bicistronic reporter RNAs established that RU5 is not an internal ribosome entry sequence and instead functions as a distinct 5′ translational enhancer.

Interestingly, translational enhancement by RU5 is greater in the context of the homologous SNV U3 promoter than in the context of heterologous CMV IE or SV40 late promoters (10-fold versus 2-fold) [62]. This difference is not attributable to changes in steady state RNA level or RNA splicing, and we speculate that the SNV U3 promoter orchestrates efficient cotranscriptional recruitment of RNA interactive factors that augment productive cytoplasmic expression of RU5-containing RNA.

Nucleocytoplasmic communication is necessary for translational enhancement by RU5

The nuclear export and translational efficiency of eukaryotic mRNAs is modulated by RNA binding proteins that shuttle between the nucleus and cytoplasm and potentially link these steps in post-transcriptional gene expression [63]. Analysis of RU5 in RNA transfection experiments and in vitro translation assays indicates that cytoplasmic proteins are not sufficient for translational enhancement by RU5 and imply that nuclear interactions between RU5 and cellular protein(s) is necessary [64]. Reporter gene assays and leptomycin B (LMB) sensitivity experiments in human 293 cells reveal that nuclear export of RU5 RNA occurs by a pathway that is independent of CRM1 and is distinct from the pathway utilized by HIV-1 RRE/Rev. RPAs demonstrated that inactivation of CRM1 by LMB enhances nuclear export of RU5-containing RNAs. Results of reporter gene assays further revealed that RU5 is sufficient to convert nonviral luc RNA from LMB sensitivity to enhancement and that LMB enhancement is sustained when RU5-containing RNA is expressed from a heterologous promoter [64].

The relationship between the RU5 nuclear export pathway and RU5-mediated translational enhancement in the cytoplasm was evaluated by competition experiments with Rev and RU5gagRRE reporter transcripts. The data indicate that upon introduction of Rev, the CRM1-dependent RRE/Rev pathway functions dominantly over the RU5 pathway to sequester RU5gagRRE reporter transcripts to the CRM1-dependent RNA export pathway. Sequestration to the RRE/Rev pathway abrogated translational enhancement by RU5. The results indicate for the first time that SNV RU5 sequences provide access to a distinct nuclear export pathway and functional linkage between a nuclear export pathway and cytoplasmic translation. The yet-to-be characterized LMB-enhanced pathway is necessary to target RU5-containing RNAs for translational enhancement in the cytoplasm.

Perspectives

Simple and complex retroviruses contain structured RNA elements that recruit cellular or viral RNA binding proteins and modulate the cytoplasmic expression of intron-containing viral and reporter RNA. HIV RRE/Rev is a prototype post-transcriptional control system that integrates multiple steps in post-transcriptional gene expression. RRE interaction with Rev in the nucleus activates the stability, nuclear export, and translational efficiency of a subset of viral transcripts. Programming of pre-mRNAs for capping, splicing and polyadenylation occurs during the transcription process and subversion by RRE/Rev of the intron removal step is expected to occur within this framework. Accordingly, the finding that Rev specifically activates cytoplasmic expression of the newly synthesized HIV RNAs indicates that RRE/Rev programs productive cytoplasmic expression of intron-containing viral RNAs during the transcription process. Together, CRM1 and adapter protein Rev commute RRE-containing RNA cargo to cytoskeletal polysomes. The potentially important role of the CRM1-dependent nuclear export pathway, which typically exports proteins and 5s rRNA, in delivery to this particular subcellular compartment remains to be determined.

RNA elements in selected simple retroviruses recruit cellular partners and augment one or more components of post-transcriptional gene expression. The CTE of MPMV interacts with cellular TAP nuclear-cytoplasmic shuttle protein and facilitates cytoplasmic accumulation of intron-containing RNA, but CTE does not modulate the translational efficiency of the RNA. The dr of ASV lacks function in the Xenopus oocyte nuclear export assay, but in avian cells functions independently of CRM1 and TAP to facilitate cytoplasmic accumulation, stability and packaging of intron-containing RNA. dr appears to compartmentalize the RNA within a cytoplasmic microenvironment that is ripe for efficient assembly of viral particles. SNV LTR sequences mark both spliced and intron-containing transcripts for a unique nuclear education that licenses nuclear export independent of CRM1 and enhances cytoplasmic association with active translational machinery. The RU5-transcripts are susceptible to reeducation by RRE/Rev, but export by the CRM-1 dependent pathway abrogates RU5-mediated translational enhancement. The data indicate that nuclear interaction between cellular factor(s) and RU5 is functionally coupled to translational efficiency in the cytoplasm. The observation that translational enhancement by RU5 exhibits promoter preference drives speculation of cotranscriptional recruitment of cellular regulatory proteins that choreograph the coupling of nuclear events with cytoplasmic expression of intron-containing RNA.