INTRODUCTION

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Retroviruses require cytoplasmic expression of unspliced RNA to produce infectious progeny. Complex retroviruses including human immunodeficiency virus type 1 (HIV) and bovine leukemia virus (BLV) exert similar posttranscriptional control by their regulatory protein and cis-acting responsive elements, Rev/Rex and RRE/RxRE, respectively. Rev and RRE are necessary for efficient transport, stability, and translation of unspliced HIV RNAs (1-3, 9-11, 13, 15, 16, 18-20, 27). Simple retroviruses lack analogous regulatory protein. However, recent studies have identified cis-acting elements in some simple retroviruses that function in conjunction with a Rev-like cellular factor(s) to modulate cytoplasmic expression of their unspliced RNA (4, 14, 23, 29, 32, 33, 38). These elements, designated constitutive/cytoplasmic transport elements or cis-acting trans-activation elements (CTEs), have been identified in Mason-Pfizer monkey virus (MPMV) (4), the related simian retrovirus 1 (SRV-1) (38), and the avian Rous sarcoma virus (RSV) (23, 29). The CTEs are structured RNA elements positioned in the 3' untranslated region (UTR) (12, 24, 32) and were identified by their ability to replace HIV Rev/RRE function in subgenomic HIV plasmids (4, 23, 38). They function to increase stability and nucleocytoplasmic transport of unspliced transcripts. The RSV CTE is proposed also to facilitate efficient processing of Gag precursor protein (29).

Recent characterization of BLV retroviral vector genomes that contain spleen necrosis virus (SNV) long terminal repeats (LTRs) revealed Rex/RxRE-independent expression of BLV structural gene vectors (5, 6). This observation indicates that SNV RNA may contain a cis-acting element that interacts with cellular Rex-like factors. SNV is an avian simple retrovirus that is unrelated to MPMV, SRV-1, or RSV and is instead related to murine leukemia virus (37).

The goal of this study was to test the hypothesis that SNV RNA contains a CTE that would interact with cellular Rev-like factors. Our analysis focused on two SNV regions: the 3' UTR that corresponds to the position of the MPMV, SRV-1, and RSV CTEs; and the LTRs, because BLV structural gene vectors that contain the SNV LTRs are Rex/RxRE independent (5). Our data eliminate the possibility that the SNV 3' UTR and 3' LTR facilitate Rev-independent gene expression and establish that the SNV 5' LTR functions in a position-dependent manner to facilitate Rev/RRE-independent expression of HIV gag RNA. The SNV 5' LTR facilitates cytoplasmic accumulation and polysome association of HIV unspliced and spliced RNAs. These data identify a novel retrovirus posttranscriptional control element located at the 5' terminus of a simple retrovirus RNA that facilitates Rev/RRE-independent expression of unspliced and spliced HIV RNAs.

	MATERIALS AND METHODS

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Plasmid construction. HIV-based plasmid pSVgagpol-rre, pSVgagpol, or pBBgagpol encodes HIV Gag, contains the simian virus 40 (SV40) promoter, and either contains RRE, lacks RRE but contains a -globin intron, or lacks RRE and a -globin intron, respectively (30). To construct derivatives of each of these plasmids, the SNV 3' UTR was excised from pKB477 on a BamHI/BglII fragment and subcloned into the BamHI site of pSVgagpol-rre, pSVgagpol, or pBBgagpol to construct pKB634, pKB636, or pKB637, respectively. The SNV LTR was excised from pKB404 on a BamHI fragment and subcloned into the BamHI site of pSVgagpol-rre, pSVgagpol, or pBBgagpol to construct pKB624, pKB628, or pKB632, respectively.

RNA preparation. Total, nuclear, or cytoplasmic RNA was prepared with Tri-Reagent or Tri-Reagent LS, respectively, as instructed by the manufacturer (Molecular Dynamics, Inc., Sunnyvale, Calif.). Transfected COS or 293 cells from two 10-cm-diameter plates or T150 flasks were harvested into phosphate-buffered saline, centrifuged at 2,000 × g for 5 min, and resuspended in 0.9 ml of cold cell lysis buffer (10 mM Tris [pH 8.3], 150 mM NaCl, 1.5 mM MgCl₂) and 0.1 ml of 5% Nonidet P-40. After thorough mixing, incubation on ice for 10 min, and centrifugation twice at 2,000 × g for 10 min at 4°C, the nuclei were treated with 1 ml of Tri-Reagent and frozen for future extraction of nuclear RNA. Following a second centrifugation step, the cytoplasmic supernatant was mixed with 3 volumes of Tri-Reagent LS, and RNA was extracted. To prepare polysomal RNA, the clarified cytoplasmic extract was supplemented with cycloheximide (50 µg/ml), RNasin (100 U/ml), and dithiothreitol (DTT; 2 mM) and layered onto a 9-ml linear gradient of 15 to 40% sucrose in 30 mM Tris (pH 7.4)-2 mM DTT-10 mM EGTA-5 mM MgCl₂ that was underlaid with 2 ml of 60% sucrose in 30 mM Tris (pH 7.4)-2 mM DTT-10 mM EDTA-5 mM MgCl₂ (26). The gradient was centrifuged 225,000 × g_max for 3.5 h at 4°C in a Beckman SW41 rotor. The EDTA in the 60% sucrose pad causes free polysomes to dissociate and sediment with membrane-bound polysomes at the 60% boundary (29). Polysomal RNA was extracted from the 60% boundary (1 ml), and nonpolysomal RNA was extracted from the upper fraction (8 ml) with Tri-Reagent. All RNA preparations were treated extensively with RQ DNase (Promega), phenol extracted, and ethanol precipitated.

RPA. Antisense runoff -³²P-labeled RNA transcripts were synthesized with MAXscript T7 RNA polymerase (Ambion) according to the manufacturer's instructions. Template pGEM(140-440) was digested with NotI, and pGAPDH was digested with NcoI. Template from pMBSVT7 was prepared by PCR amplification. The in vitro-transcribed RNAs were isolated by gel elution, and the RNase protection assays (RPAs) were performed with RPAIII (Ambion) according to the instruction manual, with some modifications. Typically, 15 µg of RNA was ethanol precipitated with 3 × 10⁵ cpm of HIV probe and 3 × 10³ cpm of glyceraldehyde-3-phosphate dehydrogenase (gapdh) probe. Samples were resuspended in 10 µl of hybridization buffer, heated at 90°C for 3 min, and hybridized at 42°C for 16 h. An RNase digestion mixture (1:100) was added to each sample (150 µl) and incubated at 37°C for 30 min. Sodium dodecyl sulfate (SDS) and proteinase K were added to final concentrations of 1% and 0.5 mg/ml, respectively; the samples were incubated at 37°C for 30 min and then subjected to extraction with phenol-chloroform and chloroform and precipitation with ethanol in the presence of 10 µg of yeast RNA. Pellets were dissolved in 6 µl of loading buffer, heated at 90°C for 3 min, and subjected to denaturing polyacrylamide gel electrophoresis (PAGE) on 5% gels. RNase protection products were visualized by PhosphorImager (Molecular Dynamics) analysis using ImageQuaNT version 4.2 (Molecular Dynamics).

	RESULTS

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SNV LTR facilitates Rev/RRE-independent production of HIV Gag. The MPMV CTE was identified originally by its ability to modulate Rev/RRE-independent expression of Gag from subgenomic HIV plasmids (4). These HIV plasmids encode gag and either contain RRE, lack RRE and contain a 3' -globin intron, or lack both RRE and a -globin intron (pSVgagpol-rre, pSVgagpol, or pBBgagpol, respectively) (35). Two SNV regions were evaluated for Rev/RRE-independent Gag production in comparison to MPMV CTE: (i) the SNV 3' UTR between env and the 3' LTR, which is analogous to the position of the previously defined CTEs (4, 24, 25, 39); and (ii) the SNV LTR, which is associated with Rex/RxRE-independent expression of BLV structural genes (5, 6). pSVgagpol-rre-MPMV and derivatives containing SNV 3' UTR (pSVgagpol-rre-3'UTR) or SNV LTR (pSVgagpol-rre-LTR) are shown in Fig. 1.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Structures of subgenomic HIV plasmid pSVgagpol-rreMPMV (5) and derivatives that contain the SNV 3' UTR and SNV LTR. SV40, SV40 late promoter; black rectangle, HIV gag-pol-vif. The SNV 3' UTR extends from the 3' 200 bp of env through the PPT.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Structures of hybrid SNV-HIV plasmids and HIV Gag production. Black lines and rectangle, HIV 5' UTR beginning at HIV U5 and extending through gag-pol, and the HIV PPT through the attL site, respectively (HIVBRU coordinates 100 to 4655 and 8662 to 8699, respectively); *, major HIV splice donor (left) and vif splice acceptor (right); arrows, sense and antisense orientation of SNV RU5. Shown at the right are representative data from 10 independent Gag ELISAs (Coulter Corp.), in which 105 293 cells were cotransfected with 2 µg of test plasmid and 0.2 µg of pEGFPN1 (Clontech) or pGL3 (Promega) reporter plasmid by the calcium phosphate protocol and maintained in DMEM with 10% fetal calf serum. Total cell proteins were harvested at 3 days posttransfection. Gag production was quantified by Gag ELISA (Coulter Corp.), and transfection efficiency was quantified as percentage of green fluorescent cells in 2,000 cells by UV microscopy or relative luciferase activity. Gag levels are normalized to transfection efficiency. <MD, less than the minimum detectable.

SNV RU5 RNA facilitates Rev/RRE-independent Gag production. To evaluate the contribution of the individual SNV LTR sequences, we analyzed a panel of hybrid SNV-HIV replacement plasmids (Fig. 2). In retrovirus DNA, the LTRs are present in two copies that are segregated into three regions: U3, R, and U5. The 5' U3 region corresponds to the promoter/enhancer, and the 3' RU5 region contains the 3' RNA processing signals. In retrovirus RNA, R sequences are repeated at both ends of the RNA transcript, U5 is unique to the 5' RNA terminus, and U3 is unique to the 3' RNA terminus. pKB504gagpol was modified by replacement of both SNV LTRs with heterologous transcriptional control sequences. The 5' SNV LTR was replaced with the CMV IE promoter and the 3' LTR was replaced with a synthetic p(A) signal to generate pYW99. Less than the minimum detectable level of Gag protein is exhibited in cells transfected with pYW99 (Fig. 2). When the 5' LTR is replaced and the 3' LTR is maintained (pYW202), low levels of Gag are observed. In contrast, when the 5' LTR is maintained and the 3' LTR is replaced (pYW100), Gag is produced at a level similar to that with pKB504gagpol. These results indicate that sequences within the 5' SNV LTR modulate Rev/RRE-independent Gag production.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Western blot immunoassay. Total cell proteins from transfected 293 cells were separated by SDS-PAGE, transferred to nitrocellulose, and reacted with polyclonal rabbit sera against HIV Gag (gift from Antonito Panganiban, University of Wisconsin). HIV Gag proteins were detected by enhanced chemiluminescence (ECL kit; Amersham). The sizes of Gag p55 and Gag p24 indicated are based on comparison with molecular weight markers (not shown).

Rev/RRE-independent Gag levels are not attributable to differences in steady-state RNA. Steady-state RNAs from pYW99, pYW100, pYW205, and HIV provirus were subjected to quantitative RPAs with an antisense RNA probe that extends across the HIV major splice donor and distinguishes unspliced and spliced HIV transcripts (Fig. 4A) (22). A gapdh probe (gift from Ing-Ming Chiu, Ohio State University) was used to normalize differences in RNA loading. Control RNA expressed from HIV^NL4-3 exhibited the HIV unspliced RNA and spliced RNAs previously characterized by McBride and Panganiban (22) (Fig. 4B). These HIV unspliced and spliced RNAs were also expressed from pYW100, pYW99, and pYW205. Interestingly, pYW100 exhibits an increased amount of spliced RNA. The size of spliced transcripts corresponds to pre-mRNAs spliced at the HIV major 5' splice donor upstream of gag and the vif splice acceptor. Results from four independent experiments indicate that steady-state gag RNA levels are 3.5 ± 1.6-fold higher for pYW99 than pYW100 and indicate that Rev/RRE-independent Gag production is not attributable to increased promoter activity or RNA stability. While a difference in splicing pattern is not observed among the RNAs, experiments were performed to more completely evaluate the possibility that the low-level Gag production from pYW205 is attributable to altered RNA splicing. The 5' RNA terminus was characterized by primer extension analysis with an antisense primer in the HIV 5' UTR. Compared to pYW99 RNA, pYW205 RNA was 1 nucleotide (nt) longer and differed in sequence at the 5' terminal 9 nt (Fig. 4C). RPAs were performed with an antisense pYW205 RNA probe that extends across SNV U3 and the HIV 5' UTR splice donor. Again similar unspliced and spliced HIV transcripts were observed from pYW205 and pYW99 (data not shown). Comparison of the protected RNAs against DNA sequence ladders confirmed that, as expected, the protected pYW99 RNAs are 10 nt shorter than the pYW205 RNAs. These data eliminate the possibility that the low level of Gag production from pYW205 is attributable to altered RNA splicing that yields new gag transcripts that either lack a 5' splice site (7) or contain an excisable intron positioned upstream of gag (17). Further experiments are necessary to explain the low-level Gag production from pYW205.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   RPA. (A) Regions of complementarity between hybrid SNV-HIV sequence and the antisense HIV RNA probes, and the protected unspliced and spliced transcripts. 5' ss, 5' splice site. SNV R and U5 RNA regions are shown in white. HIV U5 and 5' UTR (narrow line) and gag are shown in black. (B) Quantification of steady-state RNA levels by RPA. Two days posttransfection, total cellular RNA was harvested and subjected to DNase treatment. Aliquots of 15 µg were subjected to RPA with uniformly labeled antisense HIV RNA and gapdh RNA probes, PAGE, and PhosphorImager analysis. The protected RNAs are labeled. (C) Sequence comparison of the 5' termini of pYW99 and pYW205 RNA. Primer extension analysis was performed on total cell RNA from transfected cells with murine leukemia virus reverse transcriptase and primer complementary to the HIV 5' UTR. The extension products were approximately 100 bases in length and were analyzed by electrophoresis in parallel with homologous DNA sequencing reactions and PhosphorImager analysis. Sequence differences are indicated in boldface.

SNV sequences facilitate cytoplasmic accumulation of HIV RNA. To begin to address the role of SNV RU5 in the cytoplasmic accumulation of HIV RNAs, RPAs were used to analyze total and cytoplasmic RNAs from cells transfected with pYW99, pYW100, pYW205, or pYW208. The presence of the SNV RU5 correlates with a two- to fourfold increase in nucleocytoplasmic transport of both unspliced and spliced RNA (Table 2; compare pYW99 with pYW208 and pYW205 with pYW100). Moreover, the presence of the SNV LTR in pYW100 also increased the relative amount of spliced RNA significantly. The modest increase in nucleocytoplasmic transport by SNV RU5 is not sufficient to account for the significant increase in Gag production in the presence of the element. Therefore, we performed experiments to test the hypothesis that SNV RU5 enhances the polysome association of the HIV RNAs. Previous research has shown that Rev increases polysome association of Rev-dependent mRNAs (2, 10).

View larger version (81K): [in this window] [in a new window]   FIG. 5.   Polysomal RNA accumulation. RPA of nuclear, polysomal, and total RNAs. RNAs were harvested 2 days posttransfection and subjected to DNase treatment, and 5- to 10-µg aliquots were subjected to RPA with uniformly labeled antisense HIV and gapdh RNA probes, PAGE, and PhosphorImager analysis. Labels indicate the protected RNAs.

View larger version (58K): [in this window] [in a new window]   FIG. 6.   Comparison of polysomal and nonpolysomal RNA localization. RPA of nuclear, polysomal, and nonpolysomal RNAs. RNAs were harvested 2 days posttransfection and subjected to DNase treatment, and 5- to 15-µg aliquots were subjected to RPA with uniformly labeled antisense HIV and gapdh RNA probes, PAGE, and PhosphorImager analysis. Labels indicate the protected RNAs. (A) RPA of RNA from cells transfected with pSVgagpol-rre without and with pCMVRev and pSVgagpol-rre-MPMV. (B) RPA of RNA from cells transfected with pYW99 and pYW100.

	DISCUSSION

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The goal of this study was to test the hypothesis that SNV sequences contain a cis-acting element that facilitates Rev/RRE-independent expression of HIV gag RNA. SNV 3' UTR and LTR regions were analyzed in the context of HIV-based plasmids that were used previously for identification of the MPMV CTE (4). Exchange of the MPMV CTE with the SNV 3' UTR or SNV LTR indicated that these SNV regions do not function in the 3' UTR of pSVgagpol-rre to replace the function of MPMV CTE, even in the absence of RRE or a -globin intron (Table 1). Previous observation of BLV Rex/RxRE-independent gene expression in the context of hybrid SNV-BLV retrovirus vectors suggested that the SNV LTRs possess a position-dependent CTE-like function (5, 6). Consistent with this prediction, the SNV LTRs facilitated HIV Rev/RRE-independent Gag expression in the context of a hybrid SNV-HIV retrovirus vector (Table 1; Fig. 2). The observation of Rev/RRE-independent gene expression in COS, D17, and 293 cells indicates that putative cellular factors necessary for function of the SNV element are expressed in each of these cell lines (Table 1; Fig. 2). Analysis of Gag production from a panel of LTR deletion mutants indicates that the SNV element functions in a position- and orientation-dependent manner that corresponds to the 5' LTR (Fig. 2 and 3). Specifically, the SNV RU5 RNA is necessary and sufficient for efficient Rev/RRE-independent Gag production. Quantitative RPAs were used to evaluate the contributions of transcription, RNA stability, nucleocytoplasmic transport, and translation efficiency to Rev/RRE-independent Gag production. Analysis of steady-state RNA indicates that the effect of SNV RU5 is not attributable to increased steady-state gag RNA level or changes in splicing pattern, although SNV sequences do increase the amount of spliced HIV transcripts (Fig. 4B, 5, and 6B). RPAs of nuclear and cytoplasmic gag RNA indicate that SNV RU5 RNA facilitates cytoplasmic accumulation of both unspliced and spliced HIV transcripts (Table 3). Because the two- to fourfold increase in transport is insufficient to account for the significant increase in Gag production, a translational effect of the SNV sequence was investigated. Analysis of cytoplasmic localization of the RNAs indicates that the SNV 5' LTR enhances polysome association of HIV unspliced and spliced RNAs (Fig. 5 and 6; Tables 3 and 4). Control experiments with the Rev-dependent RNAs indicate that the MPMV CTE selectively facilitates polysome association of HIV unspliced transcripts (Fig. 6; Table 4).

Unexpectedly, low-level Rev/RRE-independent Gag production is detected from pYW205, which encodes the SNV U3 promoter/enhancer region alone. As expected, Gag production is not detectable from pYW99, which encodes the CMV IE promoter/enhancer alone. Comparative analysis of pYW205 and pYW99 by RPA and primer extension eliminated the possibility that pYW205 RNA lacks a 5' splice site (7) or contains an excisable intron positioned upstream of gag (17). Further experiments are necessary to understand the low-level Gag production from pYW205.

Comparison of gag RNA and protein levels from pYW205, pYW100, pYW99, and pYW208 indicates that the combination of SNV RU5 with the SNV U3 promoter/enhancer, rather than combination of SNV RU5 with the CMV promoter/enhancer, produces maximal levels of Gag. A possible explanation for the apparent synergy between the SNV U3 promoter/enhancer and RU5 is cooperative interaction between cellular factors mediated by U3 and RU5 that stimulates high-level Rev-independent Gag production. The R regions of related simple retroviruses (i.e., murine leukemia virus and chicken syncytial virus) have been shown to be important for stimulation of gene expression and the mechanisms involved in RNA processing (9).

trans activation of Gag production by Rev/RRE involves derepression of cis-acting translational repressive sequences in HIV RNA (8, 21, 25, 28) that bind cytoplasmic poly(A)-binding protein 1 (1); release of this protein is proposed to enhance polysome association and efficient translation of gag RNA by facilitating interaction between the 3' poly(A) tail and 5' 7-methylguanosine cap (1, 34). Future experiments will consider whether the SNV sequences neutralize these translational repressive sequences in the HIV RNA or supply stimulatory sequences.

In summary, SNV encodes a position- and orientation-dependent posttranscriptional control element that is distinct in location and function from the MPMV CTE. The possibility of the existence of a CTE elsewhere in the SNV genome remains. It is also possible that the MPMV LTR contains a posttranscriptional control element similar to that of SNV. The SNV element corresponds to the 5' terminus of the SNV RNA and increases cytoplasmic accumulation and polysome association of both unspliced and spliced HIV transcripts. In contrast, the MPMV CTE selectively stimulates cytoplasmic accumulation and polysome association of unspliced viral transcripts. Importantly, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for Rev/RRE-independent Gag production by SNV sequences or MPMV CTE. The data imply that a significant effect of the SNV element is enhancement of the translation efficiency of HIV gag RNA. Elucidation of SNV primary sequence and RNA structure that are necessary and sufficient for Rev/RRE-independent transport and translation will be important for identification of cellular Rev-like factors that modulate the function of this unique posttranscriptional control element.