RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

Abstract

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export.

Introduction

The HIV-1 envelope (Env) protein is encoded by the 3’ end of the HIV-1 viral RNA (vRNA) genome in a region that overlaps the Vpu open reading frame (ORF), and the Tat and Rev ORFs (Adachi et al., 1986; Li et al., 1992). Also encoded in this region is the rev responsive element (RRE) that binds to Rev (Adachi et al., 1986; Li et al., 1992). Ordinarily, the nuclear export signal (NES) of Rev binds to a complex of the cellular proteins Crm1(exportin-1, XPO-1) and Ran-GTP to facilitate the nuclear export of the HIV-1 vRNA that encodes the Gag and GagPol proteins, as well as other incompletely spliced HIV-1 RNAs (Fischer et al., 1994, 1995, 1999; Henderson and Percipalle, 1997; Yi et al., 2002; Swanson et al., 2004, 2010; Perales et al., 2005; Cullen, 2005; Groom et al., 2009; Sherer et al., 2011; Elinav et al., 2012). It is noteworthy that this exit pathway differs from that of most spliced mRNAs, which employ cellular Tap and Nxt proteins to mediate nuclear exit (Cullen, 2005). Interestingly, the Mason-Pfizer monkey virus (MPMV), a D-type retrovirus, does not encode a Rev protein, but uses a cis-active constitutive transport element (CTE) which binds Tap (NFX1) to foster the nuclear export of incompletely spliced MPMV RNAs (Pasquinelli et al., 1997; Wodrich et al., 2000; Swanson et al., 2004; Cullen, 2005).

The HIV-1 Env protein is translated from incompletely spliced 4 kb viral mRNAs to yield a glycosylated 160 kDa polyprotein (gp160) that is routed to the plasma membranes (PM) of infected cells via the vesicular transport pathway (Sodroski et al., 1986; Checkley et al., 2011). During its transit, gp160 is cleaved by furin-like proteases to yield the Env surface (SU, gp120) and transmembrane (TM, gp41) proteins, which remain associated, and assemble into trimer units (Welman et al., 2007; Checkley et al., 2011). At the PM, Env proteins may be endocytosed, by virtue of internalization motifs on the TM cytoplasmic tail (CT; Lodge et al., 1994, 1997; Egan et al., 1996; Boge et al., 1998; Courageot et al., 1999; Chan and Chen, 2006). Alternatively, Env proteins may be incorporated into assembling virus particles. Evidence suggests that assembly of wild type (WT) Env proteins into virus particles is dependent on an interaction between the Env CT and the matrix (MA) domain of the precursor Gag (PrGag) protein, but it is unclear whether this interaction is direct or indirect (Gabuzda et al., 1992; Wang et al., 1993; Yu et al., 1993; Freed and Martin, 1995a, 1995b, 1996; Mammano et al., 1995; Murakami and Freed, 2000a, 2000b; Wyma et al., 2000; Santos da Silva et al., 2013). For HIV-1 Env proteins that carry CT deletions (ΔCT), the scenerio is different. Such ΔCT proteins do not require an interaction with MA for virion incorporation (Gabuzda et al., 1992; Wang et al., 1993; Yu et al., 1993; Freed and Martin, 1995a, 1995b, 1996; Mammano et al., 1995). These observations support a model in which the long HIV-1 Env CT interferes with free diffusion into assembly sites, making it so that ΔCT proteins are passively incorporated into virions, whereas WT proteins require the CT–MA interaction (Gabuzda et al., 1992; Wang et al., 1993; Yu et al., 1993; Freed and Martin, 1995a, 1995b, 1996; Mammano et al., 1995; Murakami and Freed, 2000a, 2000b; Wyma et al., 2000; Muranyi et al., 2013; Santos da Silva et al., 2013).

During our studies on HIV-1 assembly, we observed that expression of HIV-1 Env proteins and RNAs exerted variable effects on Gag protein expression, assembly, and release. In particular, we observed a protein-mediated effect, in which WT Env protein expression reduced Gag expression and virus particle release, while ΔCT Env protein expression did not. A second effect was exerted at the RNA level. Specifically, we observed that expression of Env-encoding RNAs reduced Gag expression, processing, and release, irrespective of whether the Env protein itself was expressed. The RNA effect mapped to the RRE, and Gag proteins that were translated from RNAs that used the Tap-mediated nuclear exit pathway were unaffected. Overall our results support a model for a CT–MA interaction, and demonstrate the possibility of inhibiting HIV-1 replication via competition for RNA nuclear export.

Results

Effects of env on HIV-1 Gag

To examine interactions between the HIV-1 structural Gag proteins and the membrane-associated Env proteins, we initially utilized Gag expression vectors HIV-Luc and psPAX2 (Fig. 1; Connor et al., 1995; Zufferey et al., 1997). The former vector employs HIV-1 (Fig. 1) control elements, and therefore expression is controlled via the promoter in the long terminal repeat (LTR), and the products of the viral tat and rev genes. It is noteworthy that the env gene in this vector is non-functional due to both a frameshift and an insertion of the firefly luciferase (luc) gene (De Wet et al., 1986). In contrast to HIV-Luc, with psPAX2, the Gag and GagPol proteins are transcribed from a spliced mRNA expressed from a cytomegalovirus/chicken β actin (CAG) promoter, and most of the env gene is deleted (Zufferey et al., 1997). To complement Gag expression vectors, the HIV-1 Env protein was expressed from SIVIIIEnv (Sullivan et al., 1995) and SIVIIIΔCT, which respectively encode WT and CT-deleted Env variants (Fig. 1): in these vectors, Env is expressed in a Tat- and Rev-dependent manner.

Fig. 1.

Recombinant DNA constructs. HIV-1: shown is a map of the HIV-1 strain NL4-3 provirus with long terminal repeat (LTR) units, and the Gag, Pol Vif, Vpr (R), Vpu (U), Env, Nef, Tat and Rev open reading frames as indicated. Also depicted is the coding region for the Env cytoplasmic tail (CT), and the Rev responsive element (RRE). HIV-Luc: the HIV-Luc provirus is based on HIV-1 NL4-3, expresses Gag, Pol, Vif, Vpr, Vpu, Tat and Rev open reading frames, and carries the RRE element. It encodes the firefly luciferase gene (Luc) between NL4-3 nt 8201 of env and nt 8443 of nef. It is env- due to a frameshift at the 5’ end of env (nt 5950). HIV-gpt: the HIV-gpt provirus is based on the HIV-1 proviral clone HXB2, which is vpr-, vpu- and nef-. The HIV-gpt provirus bears a 1.2 kb deletion of the env gene (nt 6402–7620), which was replaced by a 1.1 kb fragment from pSV2gpt, encoding an SV40 promoter and origin of replication and the E. coli xanthine–guanine phosphoribosyl transferase (gpt) gene. psPAX2: psPAX2 expresses codon-optimized gag, and pol genes from spliced mRNAs driven by the a recombinant CMV enhancer/chicken β actin (CAG) promoter and employing the rabbit β globin polyadenylation signal (polyA). It carries the RRE and a fragment of the env gene, but does not express the Env protein. SVIIIEnv: this HXB2 proviral derivative carries the HIV-1 5’LTR, and encodes HXB2 sequences from an artificial SalI site at nt 5496 through the 3’ HXB2 LTR. The vector encodes vif, tat, rev and env, and carries an RRE. SVIIIΔCT: SVIIIΔCT is based on SVIIIEnv, but harbors a deletion of the Env CT, by virtue of two inserted termination codons. SVIIIStop: SVIIIStop is based on SVIIIEnv, but encodes a termination codon that truncates the Env protein after 63 codons. SVIIIpBR: the SVIIIpBR plasmid was created by replacing the HXB2 nt 5496–8470 in SVIIIEnv with a fragment from the bacterial plasmid pBR322. It does not carry the RRE and it expresses no HIV-1 genes.

To characterize the effects of WT and ΔCT Env on PrGag expression, processing, and the release of virus-like particles (VLP), cells were co-transfected with combinations of Gag and Env expression constructs. Cellular and VLP Gag levels were monitored by anti-CA immunoblotting of SDS-PAGE-fractionated samples collected at three days post-transfection. As shown in Fig. 2, co-transfection of either HIV-Luc or psPAX2 with a plasmid control (bluescript –SK; Alting-Mees and Short, 1989) gave major PrGag, p41 and CA bands in both cell and VLP samples. We also observed additional, less pronounced bands, particularly with the psPAX2 plasmid, which we infer to be Gag or GagPol processing products, as they were not present in mock-transfected or SVIIIEnv-only-transfected samples (data not shown). Surprisingly, co-transfection of HIV-Luc or psPAX2 with Env expression vectors gave significantly different results. In particular, co-transfection of HIV-Luc with either SVIIIEnv or SVIIIΔCT reduced cellular Gag levels to 18–20% control levels (Fig. 2, right panel), dramatically reduced cellular PrGag processing (Fig. 2, left panel), and almost eliminated VLP Gag signals (Fig. 2, center panel). These observations are examined in more detail in sections below. In contrast to HIV-Luc, co-transfection of psPAX2 with SVIIIΔCT appeared to have little effect on cellular and VLP Gag levels, relative to the control. However, psPAX2-directed Gag expression and VLP release were reduced to approximately 60% and 35% in SVIIIEnv cotransfections (Fig. 2, right panel). These results indicate a CT-dependent effect on Gag levels, consistent with previous observations that full-length Env protein expression may alter Gag trafficking patterns and/or may be toxic to cells (Sodroski et al., 1986; Lodge et al., 1994, 1997; Chakrabarti et al., 2002; Chan and Chen, 2006; Checkley et al., 2011; Santos da Silva et al., 2013).

Fig. 2.

Env expression effects on Gag protein expression and particle release. HEK 293 T cells (5 million on each 10 cm plate) were transfected with 12 μg of the indicated HIV-Luc or psPAX2 constructs in conjunction with 12 μg of the –SK (control), SVIIIEnv or SVIIIΔCT plasmid constructs. At 72 h post-transfection, cell lysate and virus particle samples were collected, fractionated by SDS-PAGE, and electroblotted. Gag proteins in fractionated samples were detected by immunoblotting using an antibody to HIV-1 CA. In the left two panels, PrGag, p41 and CA bands are indicated. In the rightmost panel, cellular and viral Gag bands were quantified densitometrically and normalized against the Gag levels obtained in control (plus –SK) transfections. The results represent values obtained from at least two independent experiments. Standard deviations are as indicated.

RNA level effects on Gag protein expression and release

As shown in Fig. 2, co-transfection of either SVIIIEnv or SVIIIΔCT with HIV-Luc reduced cellular PrGag expression, processing and release. To ascertain whether similar effects were exerted on wild type HIV-1, SVIIIEnv variants were co-expressed with a plasmid expressing the WT HIV-1 NL4-3 (Adachi et al., 1986) provirus. As with HIV-Luc, we found that PrGag processing was reduced relative to controls when NL4-3 was co-transfected with SVIIIEnv or SVIIIΔCT expression vectors, even though NL4-3 also encodes env (Fig. 3, left panel). Moreover, the Env expression vectors reduced both cellular total Gag levels and VLP release (Fig. 3, right panel). Similar effects also were obtained with the recombinant HIV-gpt (Fig. 1; Page et al., 1990) Gag/GagPol expression vector (data not shown). These effects were not dependent on Env protein expression, because similar effects were observed in co-transfections with the SVIIIStop plasmid, in which Env translation is terminated after 62 residues. However, the effect was not observed when the SVIIIEnv env gene was replaced by a heterologous sequence in SVIIIpBR (Fig. 3), suggesting that regulation might occur at the RNA level.

Fig. 3.

Effects of env gene variants on Gag expression, processing, and release. HEK 293 T cells (5 million on each 10 cm plate) were co-transfected with 12 μg of the NL4-3 HIV-1 proviral plasmid in conjunction with 12 μg of the –SK (control), SVIIIEnv, SVIIIΔCT, SVIIIpBR or SVIIIStop plasmids. At 72 h post-transfection, cell lysate and virus samples were collected, and subjected to SDS-PAGE and electroblotting. Gag proteins were detected by immunoblotting using a primary anti-MA antibody, and in the left two panels, PrGag and MA proteins are indicated, along with a non-specific cross-reactive cellular protein (*). For quantitation (right hand panel), cellular and viral Gag levels were determined densitometrically and normalized against the control. The results are the values obtained using duplicates of two independent transfections. Standard deviations are as shown.

Based on the above results, co-transfections of NL4-3 were performed with env gene-deleted SVIIIStop variants to localize the element(s) responsible for NL4-3 Gag protein down-regulation. Importantly, somewhat reduced cellular Gag protein levels and significantly reduced VLP-associated Gag protein levels were observed with SVIIIStop and the Δ5496–6348, Δ6348–7075, and Δ8136–8470 variants, but not with the Δ7075–8136 variant (Fig. 4). Notably, the deleted sequence in the Δ7075–8136 construct includes the RRE, suggesting a competition of SVIIIEnv RNAs with the full-length HIV-1 viral RNA (vRNA) for nuclear export mediated by the Rev-Crm1 pathway.

Fig. 4.

Mapping of the trans acting element affecting Gag protein expression and virus particle release. The left hand diagram depicts the SVIIIStop parental construct, and its four deletion derivatives, Δ5496–6348, Δ6348–7075, Δ7075–8136, and Δ8136–8474. Twelve micrograms of these plasmids or the control plasmid (–SK) were co-transfected into 5 million HEK 293 T cells on 10 cm plates along with 12 μg of the NL4-3 HIV-1 proviral plasmid, and cell and virus samples were collected at 72 h for Gag protein detection as described in Fig. 3. The bottom panels show immunoblots of cell and virus samples using a primary antibody to HIV-1 CA. Cellular and viral Gag levels were quantitated densitometrically from duplicates of two independent transfections, normalized versus the NL4-3 plus –SK control transfections, and are plotted in the graph on the upper right hand side with standard deviations as indicated. Note that in transfections with the Δ7075–8136 derivative, which lacks the RRE, cellular and viral Gag levels are approximately those of the control.

That SVIIIEnv RNAs might compete with vRNAs for nuclear export was consistent with observations that HIV-Luc, HIV-gpt, and NL4-3 HIV-1 (Fig. 1) were down-regulated, but spliced Gag messages expressed from psPAX2 were not (Fig. 2). To examine this further, we compared SVIIIStop effects on RNAs transported by different nuclear export pathways. To do so, control or SVIIIStop plasmids were co-transfected into cells with GPV-RRE, which depends on the Rev-Crm pathway (Swanson et al., 2004); or with GPV-4xCTE or GPV-RevInd, both of which utilize the Tap nuclear export pathway (Kotsopoulou et al., 2000; Swanson et al., 2004). Our results (Fig. 5) clearly showed that co-expression with the RRE-containing SVIIIStop construct markedly reduced cellular and VLP Gag levels from the GPV-RRE vector, but not from GPV-4xCTE or GPV-RevInd. These data support the notion that RRE-containing env mRNAs can compete with HIV-1 vRNAs for nuclear export.

Fig. 5.

Effects of SVIIIStop on alternative HIV-1 Gag expression constructs. Depicted in the upper panel are three alternative Gag and GagPol expression plasmids that all employ the cytomegalovirus (CMV) promoter and the SV40 polyadenylation signal (AAA), and have splice donor (SD) and splice acceptor (SA) sites. The GPV-RRE plasmid encodes HIV-1 gag, pol, vif and vpr (R) genes, and carries a copy of the RRE. GPV-4 × CTE is similar to GPV-RRE, but carries four copies of the Mason Pfizer monkey virus (MPMV) constitutive transport element (CTE) in place of the RRE. GPV-RevInd expresses codon-optimized Gag and Gag-Pol proteins from a spliced message that is Rev and RRE independent. Twelve micrograms of these plasmids were separately co-transfected onto 10 cm plates of 5 million HEK 293 T cells with either 12 μg of SVIIIStop, which is Rev+ and RRE+, or 12 μg of Δ7075–8136 (Fig. 4), which is Rev+ and RRE–. At 72 h post-transfection cell and viral Gag proteins were detected and quantified as described in Fig. 3. The lower left panels show immunoblots of cell and virus samples using a primary antibody to HIV-1 CA, and results are plotted in the lower right panel as the ratios of cellular or viral Gag levels obtained in co-transfections with RRE+ versus RRE– constructs. Results represent values obtained using duplicates of two independent transfections, and standard deviations are as shown.

In situ localization of HIV-1 vRNA

The above results suggested that the RRE-containing RNAs alter the fate of HIV-1 vRNAs after transcription. Therefore, we decided to analyze the localization of vRNAs in transfected cells. Initially, we measured total cellular full-length HIV-Luc vRNA levels by reverse transcription and polymerase chain reactions (PCR). These experiments showed that total cellular HIV-Luc vRNA levels were marginally higher (107 ± 9%; N = 4) in co-transfections with SVIIIStop (RRE+) versus SVIIIpBR (RRE–). Next, we attempted to measure nuclear and cytoplasmic vRNA levels by cell fractionation,

RNA isolation, and detection via reverse-transcription and polymerase chain reactions. However, this approach proved inadequate, possibly due to variability in fractionations. As an alternative, we employed fluorescence in situ hybridization (FISH) methods. For these experiments, cells were transfected with the HIV-Luc construct, and either SVIIIpBR or SVIIIStop, and then processed for dual detection of HIV-1 capsid proteins (by immunofluorescence) and vRNA (by FISH). The vRNAs were detected using a digoxin-labeled probe against HIV-1 pol (nt 2250–2687), followed by detection using a mouse anti-digoxin antibody, and a fluoresecently-tagged anti-mouse antibody. In this assay, because the vDNA is not denatured, labeling is specific for the vRNA (Lai et al., 2013).

In Fig. 6, images from un-transfected cells (mock) or from cells transfected with HIV-Luc plus either SVIIIpBR or SVIIIStop are shown. In each case, the CA immunofluorescence signal is in red, and the vRNA signal is green. As shown in the left hand panel, signals in un-transfected cells (mock) were below levels of detection, unless images were over-exposed (mock over-exposed), in which case cell outlines were faintly observable. For transfected cells, as expected, CA signals were cytoplasmic. Also, consistent with observed reduced Gag signals in cells co-transfected with RRE-containing SVIIIEnv variants (Figs. 2–5), CA immunofluorescence signals were fainter in SVIIIStop co-transfections than SVIIIpBR co-transfections. Importantly, vRNA localization patterns differed as well. With the SVIIIpBR co-transfections, green and yellow vRNA FISH signals were predominantly cytoplasmic. In contrast, images acquired for the SVIIIStop transfections showed a predominant nuclear accumulation of the HIV-Luc vRNA (green signal). To quantify results, over 30 images each of cells transfected with HIV-Luc and either SVIIIpBR or SVIIIStop were analyzed to determine cytoplasmic:nuclear HIV-Luc vRNA localization ratios as described in Materials and methods. The resulting values and means are graphed in Fig. 6 (right hand panel). As shown, expression of HIV-Luc in the presence of SVIIIStop, which carries the RRE, resulted in a lower cytoplasmic accumulation of the HIVLuc vRNA than did the SVIIIpBR control. Statistical analysis using an unpaired t-test indicated a significant difference in vRNA localization patterns between the two sets of samples. These results indicate that an RRE sequence present on a cotransfected plasmid reduced HIV-1 vRNA nuclear export, and are consistent with reduced cellular and VLP Gag levels observed in co-transfections with RRE-containing SVIIIEnv derivatives in Figs. 2–5.

Fig. 6.

Fluorescent in situ hybridization (FISH) analysis. HEK 293 T cells (5 million on each 10 cm plate) were co-transfected with 12 μg of HIV-Luc and 12 μg of either SVIIIpBR or SVIIIStop. At 48 h post-transfection, cells were split onto Permanox chamber slides (Nalge Nunc International) at a 1:20 ratio and incubated for 24 h and then processed for FISH analyses as described in Materials and methods. For detection of HIV-1 Gag, a primary anti-CA antibody was used in conjunction with a secondary Alexa Fluor 568-conjugated (red) anti-mouse antibody. To detect the viral RNA, a digoxin-labeled probe against HIV-1 pol (nt 2250–2687) was employed, and detected with a primary anti-digoxin antibody, and a secondary Alexa Fluor 488-conjugated (green) antibody. In the far left panels, images from un-transfected cells (mock) are shown, using either standard imaging conditions, or with double the standard brightness value (mock over-exposed) to show the faint outlines of the cells. Also shown are examples of cells transfected with HIV-Luc plus SVIIIpBR or SVIIIStop. Note the predominant nuclear vRNA (green) staining for the SVIIIStop versus the SVIIIpBR transfections. In the right hand panel, individual cells were scored for cytoplasmic versus nuclear viral RNA (vRNA) FISH signals, and cytoplasmic/nuclear ratios so obtained were graphed on the scatter plot, along with the group mean values. The unpaired t-test value (p=0.0001) indicates a significant difference in the cytoplasmic versus nuclear ratios in cells co-transfected with the SVIIIpBR (RRE–) versus SVIIIStop (RRE+) plasmids.

Effects of Gag levels on VLP release

In the course of our studies, we observed considerably greater effects of RRE competition on VLP release levels than on cellular Gag levels (Figs. 2–5). Potentially, such results could indicate that proteins translated from HIV-1 vRNAs in the presence or absence of export competitor RNAs may have qualitatively different fates (Swanson et al., 2004). Alternatively, the more drastic reductions in VLP release levels than cellular Gag levels in the presence of RRE-containing competitors may simply reflect the possibility that virus release is not linearly proportional to intracellular Gag levels (Sherer et al., 2011). To test these alternatives, cells were transfected with decreasing amounts of HIV-1 NL4-3 plasmid, after which cellular and VLP-associated Gag levels were determined. As depicted in Fig. 7, as much as an eight-fold reduction in transfected NL4-3 plasmid only reduced cellular Gag levels 2- to 3-fold. In contrast, a two-fold reduction in transfected plasmid reduced VLP-associated Gag levels approximately three-fold, and an eightfold reduction of transfected plasmid yielded at least a 20-fold reduction in VLP Gag signals (Fig. 7). These data are compatible with the notion that the observed reductions of VLP Gag are a direct consequence of reduced cellular Gag levels, but do not rule out the possibility that perturbation of vRNA nuclear export pathways may confer qualitatively different Gag protein fates.

Fig. 7.

Comparison of cellular and viral Gag protein levels. Various amounts of the HIV-1 NL4-3 proviral plasmid (16, 8, 4 and 2 μg) were used to transfect 10 cm plates of 5 million HEK 293 T cells in parallel. For each transfection, the total amount of DNA transfected was normalized to 24 μg by the addition of –SK carrier plasmid. At 72 h post-transfection, cell and viral samples were subjected to electrophoresis and immunoblotting with an anti-CA antibody (left hand panels). Gag levels in cell and viral samples from two independent experiments were quantified as described in Materials and methods, and results are plotted as percentages of the maximum cellular or viral Gag levels obtained.

Discussion

The env coding region at the 3’ end of the HIV-1 genome impacts virus particle release at the protein level and at the RNA level. Previous work has demonstrated an interaction between HIV-1 Env and the MA domain of PrGag (Gabuzda et al., 1992; Wang et al., 1993; Yu et al., 1993; Freed and Martin, 1995a, 1995b, 1996; Mammano et al., 1995; Murakami and Freed, 2000a, 2000b; Wyma et al., 2000; Santos da Silva et al., 2013). A number of studies have shown that incorporation of WT Env proteins into virus particles requires a direct or indirect interaction with MA, supporting a model in which PrGag MA domains usher Env proteins into virions through an association with the Env CT tail (Gabuzda et al., 1992; Wang et al., 1993; Yu et al., 1993; Freed and Martin, 1995a, 1995b, 1996; Mammano et al., 1995; Murakami and Freed, 2000a, 2000b; Wyma et al., 2000; Checkley et al., 2011; Santos da Silva et al., 2013). It also is noteworthy that HIV-1 Env influences the sub-cellular localization of PrGag proteins, and that this effect is dependent, at least in part, on a YSPL endocytic motif on the Env CT (Lodge et al., 1994, 1997; Egan et al., 1996; Boge et al., 1998; Courageot et al., 1999; Chan and Chen, 2006). The situation is different with ΔCT HIV-1 Env proteins, which appear to be passively incorporated into HIV-1 particles, presumably because they localize to the proximity of assembly sites (Freed and Martin, 1995a, 1995b; Mammano et al., 1995; Murakami and Freed, 2000a, 2000b; Checkley et al., 2011; Santos da Silva et al., 2013). Our results with co-transfected psPAX2 and WT or ΔCT Env protein expression vectors clearly show a CT-dependent effect of the HIV-1 Env protein on Gag protein cellular levels, localization, and release (Fig. 2). Specifically, expression of the psPAX2 vector with WT SVIIIEnv, but not SVIIIΔCT reduced cellular levels of PrGag, as well as virus release levels (Fig. 2). These data are consistent with previous observations that WT Env directs PrGag localization and stability either directly or indirectly (Sodroski et al., 1986; Lodge et al., 1994, 1997; Chakrabarti et al., 2002; Chan and Chen, 2006; Checkley et al., 2011; Santos da Silva et al., 2013).

Our data also show that expression of HIV-1 Env RNAs interfered with Gag expression from HIV-1 proviral constructs including HIV-Luc (Fig. 2), HIV-gpt (data not shown), and the NL4-3 WT strain (Figs. 3 and 4). We observed effects that ranged from 30% to five-fold reductions in cellular Gag levels, with much more significant decreases in PrGag processing and Gag protein release (Figs. 2–4). The effects of Env RNAs on PrGag were independent of the CT (SVIIIΔCT; Figs. 2 and 3) and even Env protein translation (SVIIIStop; Figs. 3 and 4), and mapped to the RRE (Δ7075–8136, Fig. 4). That expression of RRE-containing transcripts in trans appeared to regulate PrGag levels suggested a competition for the Rev-mediated nuclear export pathway might be responsible for these results (Fischer et al., 1994, 1995, 1999; Henderson and Percipalle, 1997; Yi et al., 2002; Swanson et al., 2004, 2010; Perales et al., 2005; Cullen, 2005; Groom et al., 2009; Sherer et al., 2011; Elinav et al., 2012). Our in situ localization of HIV-Luc vRNAs in cells co-transfected with SVIIIStop versus SVIIIpBR supports this interpretation (Fig. 6). Specifically, relative to SVIIIpBR, which does not encode an RRE, co-transfection of RRE-encoding SVIIIStop with HIV-Luc reduced nuclear export of HIV-Luc vRNAs, and Gag expression levels (Fig. 6). Consistent with the RRE competition model, HIV-1 Env RNA expression did not appreciably affect Gag levels from GPV-4xCTE RNAs (Fig. 5), which employ MPMV CTEs to direct vRNA nuclear export via the Tap pathway (Pasquinelli et al., 1997; Wodrich et al., 2000; Swanson et al., 2004; Cullen, 2005), or from spliced psPAX2 or GPV-RevInd RNAs (Figs. 2 and 5), which also presumably exit the nucleus via the Tap pathway (Swanson et al., 2004).

At this point, it is unclear as to which factor in the Rev-Crm1 nuclear export pathway might be limiting, and thus responsible for the observed competition in RRE-mediated cytoplasmic delivery of vRNAs. Conceivably, the limiting factor could be Rev, the cellular factors Crm1 or Ran-GTP, or other known (RanBP3, DDX3, RanBP2, NUP358) or unknown members of the pathway (Fischer et al., 1994, 1995, 1999; Henderson and Percipalle, 1997; Yi et al., 2002; Swanson et al., 2004, 2010; Perales et al., 2005; Cullen, 2005; Groom et al., 2009; Sherer et al., 2011; Elinav et al., 2012). It is also unclear as to whether the drastic reductions we have observed in PrGag processing and release directly reflect cellular PrGag levels, or whether other nuclear export pathway-specific effects are at play. Our data (Fig. 7) and the data of others (Sherer et al., 2011) suggest that PrGag release levels are not linearly proportional to cellular PrGag levels, but that this is a pathway-independent phenomenon. Nevertheless, it remains possible that variations in nuclear exit strategies, or effects on host factors impacting stabilities, modifications, or trafficking may influence the fates of messages and their translation products (Swanson et al., 2004; Cullen, 2005; Sherer et al., 2009; Jin et al., 2009). In either case, our data strongly support the idea that inhibition of HIV-1 vRNA nuclear export is a potentially viable anti-viral approach (Mautino and Morgan, 2002; Ward et al., 2009; Wong et al., 2013).

Materials and methods

Recombinant DNA constructs

The parental HIV-Luc (pNL-LucE-R ) (Connor et al., 1995) and HIV-gpt (Page et al., 1990) constructs+ were kindly provided by Nathaniel Landau. The HIV-1 NL4-3 molecular clone (Adachi et al., 1986), and psPAX2 (pCMVdeltaR8.91) and SVIIIEnv vectors (Zufferey et al., 1997; Sullivan et al., 1995) were obtained from the NIH AIDS reagent program. GVP-RRE, GPV-4xCTE and GPVRevInd (Kotsopoulou et al., 2000) were kindly provided by Michael Malim (Swanson et al., 2004). SVIIIΔCT was created by cloning the SalI–BamHI band (nt 5785–8465) from pNL4-3ΔCT144 (Freed and Martin 1995a; 1995b), in which the C-terminal 144 amino acids from gp41 were removed, into the SalI–BamHI sites of SVIIIEnv: the Env protein is thus truncated at the beginning of the CT at residues SIVNR VRQGY X. SVIIIStop was created by a DNA insertion at the NdeI site of env (HXB2 nt 6401). The sequence at the NdeI site (shown in italics) is GCA TAT GAT TAA TAT CTA GAG CAT ATT ATG, where the TAA stop codon that truncates Env after 63 codons is indicated in bold. SVIIIpBR was created by replacing the SalI– BamHI fragment (nt 3610–6130) of the SVIIIEnv (corresponding to HXB2 nt 5496–8470) by the SalI–BamHI fragment of the pBR322 plasmid (nt 651–375; Bolivar et al. 1977). The Δ5496–6348, Δ6348–7075, Δ7075–8136 and Δ8136–8470 vectors represent deletions of SVIII at the indicated HXB2 sequences and were generated by standard molecular biology procedures. In Δ5496–6348, the SalI–KpnI fragment was replaced by an oligo yielding the sequence GTC GAC CCC GGG GGT ACC. In Δ6348–7075, the KpnI– PvuII fragment was replaced by an oligo yielding the sequence GGT ACC CCC GGG CAG CTG. In Δ7075–8136, the PvuII–HindIII fragment was replaced by an oligo yielding the sequence CAG CTG CCC GGG CAA GCT T. In this vector the RRE sequence is no longer present. In Δ8136–8470, the HindIII–BamHI fragment was replaced by an oligo yielding the sequence AAG CTT CCC GGG GGA TCC.

Cell culture, transfections and immunoblotting

HEK 293 T (DuBridge et al., 1987) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.4), penicillin, and streptomycin at 37 °C and 5% CO₂. Analysis of cellular protein expression and virus particle assembly and release was performed at 48–72 h after calcium phosphate transfections of HEK 293 T cells (5 million cells on 10 cm plates) by using a total of 24 μg plasmid DNA constructs as described previously (Scholz et al., 2005, 2008; Noviello et al., 2011; López et al., 2013). All such experiments were performed at least twice with at least two different plasmid DNA preparations. Virus samples were collected by filtering media supernatants through 0.45 μm filters, followed by pelleting through 20% sucrose cushions in phosphate-buffered saline (9.5 mM sodium potassium phosphate [pH 7.4], 137 mM NaCl, 2.7 mM KCl) for 2 h at 100,000g at 4 °C (Scholz et al., 2005, 2008; Noviello et al., 2011; López et al., 2013). Pelleted viruses were carefully resuspended in cold PBS and stored at –80 °C. Cells were washed in cold PBS and then lysed in cold IPB (20 mM Tris–hydrochloride [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1.0% Triton X-100, 0.02% sodium azide). To remove insoluble debris, lysates were centrifuged at 4 °C for 15 min at 13,000g. Supernatants were collected and stored at –80 °C prior to use. Both virus and cell samples were mixed with equal volumes of 2× sample loading buffer (12.5 mM Tris–HCl [pH 6.8], 2% SDS, 20% glycerol, 0.25% bromphenol blue, 10% β-mercaptoethanol), fractionated via 10–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (BioRad Laboratories). For HIV-1 Gag protein immunoblotting, a mouse anti-CA monoclonal antibody Hy183 (kindly provided by Bruce Chesebro) was used at a 1:10 dilution (from culture media) or an anti-MA antibody (Capricorn Products; HIV-018-48170) was used at a 1:800 dilution. Detection of Gag proteins was achieved using a secondary alkaline phosphatase-conjugated anti-mouse antibody (Promega) at a 1:10,000 dilution, and subsequent color reactions for visualization of antibody-bound bands with nitroblue tetrazolium plus 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris–hydro-chloride (pH 9.5), 100 mM NaCl, and 5 mM MgCl₂. Cell and virus Gag levels were quantified densitometrically using NIH ImageJ software.

Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) experiments were performed on transfected HEK 293 T cells at 72 h post-transfection. All the steps were performed using RNase free solutions containing 2 mM vanadyl ribonucleoside (VRC). The DNA pol gene probe was prepared by asymmetric-PCR using Pol2687 reverse primer 5’-TTTCC CTTCC TTTTC CATCT CTG-3’, Gag2250 forward primer, 5’-TTCCC TCAGG TCACT CTTTG GC-3’, HIV-gpt plasmid as template, and a PCR-DIG Probe Synthesis Kit (Roche Diagnostics). For FISH incubations, transfected HEK 293 T cells were split 24 h post transfection onto Nunc Lab-Tek Chamber Slide system slides, previously treated with 0.3 ml polylysine (0.1 mg/ml; Sigma) for 30 min at 37 °C, and washed and incubated in complete media (DMEM, 10% FBS). At 72 h post-transfection, cells were washed twice with 0.5 ml PBS, and then fixed with 5% formaldehyde, 2 mM VRC and 2% sucrose in PBS for 20 min at room temperature. After three washes with PBS, cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were then pre-incubated with hybridization solution (Sigma H7140) containing the following constituents: 100 μg/ml DNA, 5 × SSC (1 × SSC= 150 mM NaCl, 15 sodium citrate), 1 × Denhardt's solution (0.02% BSA, 0.02% ficoll 400, 0.02% polyvinylpyrrolidone), 50% formamide, 5 mM dithiothreotol (DTT), 2 mM VRC. This pre-incubation step was for 30 min at 45 °C, and the hybridization solution had previously been heated to 75 °C for 10 min for denaturation. After preincubations, hybridizations were performed with 0.2 ml of 400 ng/ml probe (previously denatured at 75 °C for 10 min, and then quenched on ice for 3 min) in hybridization solution overnight at 50 °C in humidified chambers. Cells next were washed twice, 5 min each, with 45% formamide, 2 × SSC, 2 mM VRC at 45 °C; once with 2 × SSC, 2 mM VRC at 45 °C for 15 min, and once with 2 × SSC, 2 mM VRC at room temperature for 10 min. Cells then were incubated with a mouse anti-digoxin monoclonal antibody (IgG fraction; Jackson Immuno Research Laboratories, Inc.) at a 1:200 dilution in 4 × SSC, 2 mM VRC and 1% BSA for 2 h at room temperature. After three washes, secondary antibody incubation was for 1 h at room temperature using an Alexa Fluor 488-conjugated goat anti-mouse antibody (Life Technologies) at a 1:1000 dilution. After this step, cells were washed three times with complete media, and incubated with the undiluted anti-CA Hy183 monoclonal antibody for 2 h at room temperature, followed by three complete media washes, and a 1 h, room temperature secondary antibody incubation, using Alexa Fluor 568-conjugated goat anti-mouse IgG (Life Technologies) at a 1:1000 dilution in complete media. Following these incubations, the cells were washed twice with complete media followed by two washes in PBS. Cells then were incubated in cold methanol (100%) for 10 min at room temperature, followed by one wash in PBS. Coverslips so treated were mounted using FluoroGuard antifade reagent (Bio-Rad Laboratories) and imaged on a Zeiss Axiovert 200 M deconvolution microscope equipped with Chroma filters 41,001 (excitation 480 nm, emission 535 nm) and 41,004 (excitation 560, emission 645 nm). Images were acquired using a 40x objective lens on an AxioCam MRm Zeiss camera. Stacks of images were analyzed using the AxioVision Zeiss software. Alternatively, images were collected on a Zeiss Axio Observer Z1 inverted microscope as described in the immunofluorescence section above. For fluorescence intensity quantitation of the vRNA signals in acquired images, Image J software was utilized. Briefly, nuclear and cellular boundaries were selected by hand, and mean brightness values for vRNA FISH signals were quantified to obtain nuclear and total cell areas and mean fluorescence brightness values. Cytoplasmic brightness values were determined by the formula cytoplasmic mean brightness = [(total cell area × mean cell brightness) – (nuclear area mean nuclear brightness)]/(total area – nuclear area). For statistical purposes, data obtained from these images were plotted using Prism5 software. Differences were calculated to be significant to a P value of <0.0001, using the unpaired two-tailed t test calculation function of Prism5.