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Journal of Virology, October 2007, p. 10515-10523, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00947-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vpr Is Required for Efficient Nef Expression from Unintegrated Human Immunodeficiency Virus Type 1 DNA

Betty Poon, Michael A. Chang, and Irvin S. Y. Chen^*

Departments of Microbiology, Immunology and Molecular Genetics, and Medicine, David Geffen School of Medicine at UCLA, UCLA AIDS Institute and Jonsson Comprehensive Cancer Center, Los Angeles, California

Received 2 May 2007/ Accepted 13 July 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Unintegrated human immunodeficiency virus (HIV) DNA are viral DNA products formed naturally during HIV replication. While the integrated proviral DNA form is transcriptionally active and results in productive infection, unintegrated DNA is also capable of expression of viral RNA and proteins. Previously, we showed that HIV Vpr enhances expression from integrase-defective HIV. Here we show that Vpr activation of expression is partially dependent upon the presence of a transcriptionally active HIV promoter and results in increased transcription of unspliced gag and spliced nef viral RNA. While Tat is detectable during infection with integrase-defective HIV, Tat levels are not affected by the presence of Vpr. Mutation studies reveal that Tat is dispensable for the Vpr-mediated enhancement of expression from unintegrated DNA. We find that virion-associated Vpr is sufficient for Nef expression from unintegrated viral DNA, resulting in the efficient downregulation of CD4 from the surface of infected cells. These results provide a mechanism by which Nef expression from unintegrated HIV type 1 DNA expression occurs.

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Human immunodeficiency virus type 1 (HIV-1) Vpr is a virion-associated protein with multiple critical roles in viral replication and pathogenesis. A role for Vpr in AIDS pathogenesis was demonstrated in experimentally infected rhesus macaques, in which simian immunodeficiency virus carrying a mutated vpr gene reverted to the wild type in some animals and was associated with a more rapid progression to disease (20, 25, 33). Both the de novo-expressed and virion-associated forms of Vpr induce cell cycle arrest in mammalian cells at the G₂/M phase. Vpr-induced G₂ arrest is characterized by inactivation of Cdc kinase and requires the activity of protein phosphatases PP2A and Cdc25. The induction of G₂ arrest by Vpr may directly or indirectly contribute to increased HIV-1 expression in both dividing and nondividing cells through increased transcriptional activity of the HIV long terminal repeat (LTR) (22, 50). Vpr also possesses transcriptional activity and can weakly transactivate the HIV-1 LTR and other heterologous promoters (14). Mutation analysis revealed that Vpr transactivation of HIV-1 is mediated through cis-acting elements within the viral LTR, and in vitro binding studies show interaction between Vpr and Sp1, TF-IIB, and cyclin T1 (3, 44, 54, 55). Another key function of Vpr is facilitating the efficient infection of nondividing cells such as macrophages, where Vpr participates in the nuclear import of the preintegration complex, thus contributing to the establishment of a long-lasting virus reservoir and aiding in subsequent viral spread to lymphoid organs and T-helper lymphocytes (17, 24). Vpr induces apoptosis in HIV-infected primary lymphocytes (18, 26, 36, 47, 49), thus possibly contributing to immune dysregulation and CD4⁺ T-cell depletion.

Infection by retroviruses, including HIV-1, results in the reverse transcription of the viral genome and the formation of double-stranded DNA, which is subsequently imported into the nucleus and integrated into the host chromatin. In addition to the integrated forms of HIV-1 DNA, HIV-1 infection results in nonintegrated viral DNA including linear and one and two LTR circular forms. Abundant levels of unintegrated HIV-1 DNA can be detected in vivo (10, 13, 38, 46, 51), and unintegrated DNA is the most prevalent form of DNA (2 log more than integrated DNA) in resting and activated CD4⁺ T cells in vivo (13). Integration of HIV is usually required for efficient viral replication and expression, although recent reports suggest that transcription from nonintegrated HIV DNA appears to be a normal early step in HIV replication. Unintegrated viral DNA, formed by integrase-defective viruses or from integration-competent virus in the presence of integrase inhibitors, has transcriptional activity in vitro, resulting in the expression of viral RNA and proteins (9, 21, 57, 58). Infection of MAGI cells with integrase-defective viruses resulted in ß-galactosidase expression, indicating the presence of Tat (19, 56). Both nef mRNA and protein were evident upon infection of primary T cells and T-cell lines (21, 57, 58). Previously, we showed that Vpr is essential for the efficient expression from unintegrated HIV-1 DNA, resulting in a marked increased in expression upon infection with integration-defective HIV-1 expressing the luciferase gene in place of the nef gene (40).

Here, we characterize the potential mechanisms and consequences of Vpr-mediated expression from unintegrated HIV-1 DNA. The effect of Vpr on unintegrated DNA is partially mediated by sequences present within the LTR. Vpr increases the levels of unspliced gag/pol and spliced nef transcripts but has no effect on the level of spliced tat/rev transcripts. Vpr-induced expression from unintegrated DNA is independent of Tat function. In the presence of Vpr, there is increased expression of Nef protein, resulting in the efficient down-regulation of CD4 from infected cell surfaces. Therefore, Vpr acts at a transcriptional and nontranscriptional level on unintegrated HIV DNA. In addition, Vpr, and not Tat, is a major regulator of unintegrated DNA expression, and Vpr is required for the expression of HIV-1 Nef from unintegrated HIV-1 DNA.

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DNA constructs. The nucleotide position numbers used to describe the vector construction start at the 5' end of HIV_NL4-3 (1). The NF-B and Sp1 consensus binding sites within the 3' LTR of NLlucDA (40) were altered by site-directed mutagenesis using primers (altered nucleotides are underlined) 5'-GAGCTTGCTACAATCTACTTTCCGCTGTCTACTTTCCAGGG-3' (nucleotides 9412 to 9452) and 5'-CTTTCCAGGGATTCGTGGCCTGTTCGGGACTGGTTAGTGGCGTG-3' (nucleotides 9443 to 9486), respectively, resulting in NLlucDANF-B, NLlucDASp1, and NLlucDANF-B/Sp1. The self-inactivating HIV-based vector lacking the internal promoter sin18 PGK-luc was derived from pRRLhPGKeGFPsin (59) by deleting the phosphoglycerate kinase (PGK) promoter and replacing the enhanced green fluorescent protein (eGFP) gene with the luciferase gene from pGL3 Basic (Promega). The murine leukemia virus (MLV)-based vector expressing luciferase from an internal HIV-1 LTR, MLV HIV LTR luc, contains the LTR-luciferase cassette from pGL3 LTR-luc inserted into SR-MSV sin (4). The HIV-1 expressing all HIV genes except that of the envelope was constructed by deleting envelope sequences 7032 to 7612 from HIV_NL4-3, resulting in pNL Bgl. HIV-1 containing truncated Vpr (Vpr^–) (pNL Bgl VprX) or mutated integrase (pNL Bgl D64E) or mutations in both Vpr and integrase (pNL Bgl VprX D64E) was constructed by cloning the vpr or the integrase gene from NLlucBgl VprX, NLlucBgl D64E, or NLlucBgl VprX D64E (40), respectively, into pNL Bgl. The tat-deleted, vpr-deleted constructs NLlucBgl VprX TatX1 and NLlucBgl VprX TatX1 D64E contain the insertion of a linker sequence (5'-TTAGGTCTAGACCCGGGCGGCCGATCGATCC-3') into the Sau1 site (nucleotide 5954) of NLlucBgl VprX and NLlucBgl VprX D64E, resulting in a frameshift within the tat gene. The HIV LTR-luciferase expression construct pGL3 LTR-luc was constructed by cloning the 3' LTR from NLlucDA into pGL3 Basic (Promega). The integrase-defective packaging plasmid pCMVR8.2DVpr D64E and the lentiviral vectors pHR'CMV-eGFP and pHR'CMV-Vpr have been previously described (5, 40, 48). A point mutation (D125E) was introduced by site-directed mutagenesis into the catalytic triad motif of integrase in SVpsi-env-MLV (32) and used to produce integrase-defective MLV virions.

Virus production. Vesicular stomatitis virus G protein (VSV-G)-pseudotyped viruses were produced by calcium phosphate-mediated transfection of 293T cells, as described previously (41). Cells (2 x 10⁷) were transfected with 5 µg of pHCMVG and 12.5 µg of full-length HIV plasmids or, for the production of retroviral vectors, 5 µg of pHCMVG, 12.5 µg of packaging plasmid, and 12.5 µg of the appropriate retroviral vector. Virus stocks of HIV IN⁺/Vpr⁺, HIV D64E/Vpr⁺, HIV IN⁺/Vpr^–, and HIV D64E/Vpr^– were generated using NLBgl, NLBgl D64E, NLBgl VprX, and NLBgl VprX D64E, respectively. The tat-deleted, vpr-deleted Vpr^–/Tat^–/IN⁺ and Vpr^–/Tat^–/D64E viruses were produced using NLlucBgl VprX TatX1 and NLlucBgl VprX^–TatX1 D64E, respectively. Lentiviruses HR-eGFP and HR-Vpr were produced using pCMVDR8.2DVpr with pHR'CMV-eGFP or pHR'CMV-Vpr, respectively. The integration-defective lentiviral vectors were produced using pCMVR8.2DVpr D64E and NLlucDA, NLlucDANF-B, NLlucDASp1, NLlucDANF-B/Sp1, and sin18 PGK-luc. The integration-defective MLV vectors were produced using SVpsi-env-MLV D125E and MLV HIV LTR luc. Vpr was incorporated into HIV-1 virions using NLBgl VprX or NLBgl VprX D64E and BSVpr (39).

Culture supernatants were collected and concentrated as previously described (41). Multiplicity of infection (MOI) was determined by infecting 2 x 10⁴ HeLa cells with HR-eGFP and using flow cytometry to analyze for eGFP positivity at day 2 postinfection. Twenty nanograms and 200 ng of p24 antigen of VSV-G-pseudotyped viruses resulted in MOIs of 0.1 and 1.0, respectively.

Cell culture. 293T cells, HeLa cells, and HeLa-tat cells were maintained in Dulbecco's minimal essential medium with 10% calf serum. SupT1 cells were maintained in Iscove's medium with 10% fetal calf serum. Cells were infected with VSV-G-pseudotyped viruses for 4 h at 37°C in the presence of 10 µg/ml polybrene. HeLa cells were transfected using Lipofectin Plus (Invitrogen), according to the manufacturer's instructions.

Cells were harvested 40 h postinfection, lysed in 50 to 100 µl of 1x passive lysis buffer (Promega), and analyzed for luciferase activity. Luciferase activity was measured using a Monolight 2010 luminometer machine and normalized to relative luciferase units/µg total protein after protein levels were determined using the Bradford assay (7a). Each sample was analyzed in duplicate or triplicate, and the average deviations were calculated. The background luciferase detection level was 0.3 x 10² relative luciferase units/µg.

Infected SupT1 cells were immunostained with fluorescein isothiocyanate-conjugated anti-human CD4 antibody (clone SK3; BD Biosciences) according to the manufacturer's recommendations, and 5,000 events per sample were acquired on a FACScan II (Becton-Dickinson). The percentages of CD4-positive and -negative cells were analyzed using Cell Quest software (Becton-Dickinson).

Western blotting analysis. At 48 h postinfection, cells (1 x 10⁵) were harvested and lysed in radioimmunoprecipitation assay buffer in the presence of protease inhibitors (Sigma-Aldrich). The protein concentration was determined by the Bradford assay (7a), and equivalent amounts of total protein (10 µg) were heated at 100°C for 5 min, separated by 17% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to Immobilon P nylon membranes (Amersham). Membranes were incubated with the following reagents (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH): HIV Nef monoclonal antibody AG11 from James Hoxie, followed by horseradish peroxidase-linked anti-mouse secondary antibody (Santa Cruz Biotech), and visualized using ECL plus (Amersham).

RT-PCR. Total RNA from infected SupT1 cells was isolated using a QIAGEN RNeasy extraction kit and treated with DNase I (RNase-free DNase set; QIAGEN) according to the manufacturer's instructions. Quantification of mRNA was performed using an ABI PRISM7700 (Applied Biosystems) with a OneStep reverse transcription (RT)-PCR kit (QIAGEN). The primer pairs and cycling conditions for the HIV PCR amplifications have been described previously (6, 8, 53). Human ß-2 microglobulin primers have been described previously (8). In vitro-transcribed standards for the quantification of ß-2 microglobulin and full-length, tat/rev, and nef transcripts were generated using a MegaScript T7 kit (Ambion).

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Vpr transactivation of integrase-defective HIV is partially dependent on cis-enhancer sequences. We previously showed that Vpr enhances expression from integrase-defective HIV-1 and that the enhancement is dependent on the presence of the HIV-1 LTR. The ability of Vpr to transactivate the HIV-1 LTR in transient-transfection assays has been attributed to cis-acting elements within the U3 region of the HIV-1 LTR, including Sp1, NF-B, and glucocorticoid response element binding sites (3, 54, 55). We utilized an integration-defective HIV-1-based vector in which luciferase was transcribed off the HIV-1 LTR to explore the contributions of these previously reported transcriptional elements. We first examined the effect of mutation of the NF-B or Sp1 binding site on the ability of Vpr to increase expression from unintegrated DNA (Fig. 1). HeLa cells were coinfected with the HIV-1 vector expressing luciferase and a lentiviral vector expressing either eGFP or Vpr. In the context of an intact HIV LTR, coinfection with Vpr increased the expression of luciferase 35-fold relative to that of coinfection with eGFP. Loss of either the NF-B or the Sp1 binding site (NF-B and Sp1, respectively) resulted in a 15-fold or 10-fold increase in luciferase, respectively, in the presence of Vpr. When both the NF-B and the Sp1 binding sites were mutated (NF-B/Sp1), coinfection with HR-Vpr resulted in an eightfold increase in luciferase activity. We conclude that the NF-B and Sp1 binding sites are important for Vpr-mediated expression from integration-defective HIV-1, as loss of these elements resulted in a lower enhancement of luciferase expression by Vpr. However, additional sequences within HIV-1 are likely involved as Vpr can still enhance expression, albeit at weaker levels, when both the Sp1 and NF-B sites are mutated.

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FIG. 1. Vpr transactivation of integration-defective HIV-1 is partially dependent on cis-enhancer sequences. HeLa cells (2 x 104 cells) were coinfected with integrase-defective viral vectors as indicated and HR-eGFP or HR-Vpr (200 ng of p24 antigen). HIV vector indicates infection with HIV-based vector expressing luciferase under the control of an intact HIV-1 LTR (HIV LTR) (MOI of 0.1, using 25 ng of p24 antigen), an HIV LTR with a mutation in the NF-B (NF-B) or the Sp1 (Sp1) or both the NF-B and Sp1 (NF-B/SP1) binding sites, or an HIV LTR containing a 400-bp deletion in U3 (sin18) (MOI of 1.0, using 200 ng of p24 antigen). MLV vector indicates infection with MLV-based vector expressing luciferase under the control of an internal HIV LTR. Luciferase activity was analyzed at day 2 postinfection and normalized by micrograms of total protein. The induction change (n-fold) by Vpr is calculated as the level of luciferase activity in the HR-Vpr-infected cultures relative to that of the HR-eGFP-infected cells, shown as the mean ± standard deviation of three separate wells. This is a representative experiment from five independent experiments.

To delineate whether these additional sequences are classical transcriptional elements that are located within the HIV LTR, we next examined the ability of Vpr to transactivate a self-inactivating (SIN) integrase-defective HIV-based vector with extensive deletions in the HIV-1 LTR engineered to completely abrogate HIV-1 LTR-driven transcription (sin18). This vector lacks 400 of the 455 bases of the U3 region, including the NF-B, NFATc, and Sp1 binding sites and the TATA box (59). There were low but detectable levels of luciferase upon infection of HeLa cells with the sin18 vector lacking an internal promoter (data not shown), in agreement with previously published reports of SIN vectors retaining some transcriptional activity due to residual promoter activity from elements downstream of region U5 in the LTR (34). Coinfection with HR-Vpr resulted in a 12-fold increase in luciferase expression from the integration-defective sin18 lentiviral vector, lower than the 35-fold increase seen from the integrase-defective virus with an intact LTR and similar to the increase seen with the NF-B/Sp1 vectors. In three independent experiments, there was a consistent three- to fourfold reduction in the extent of Vpr-mediated transactivation of the sin18 vector relative to that of the HIV vector containing an intact LTR. Thus, Vpr can still enhance HIV-1 expression, even upon deletion of virtually all upstream sequences present in the LTR.

We confirmed the requirement for non-LTR elements by studying the ability of Vpr to transactivate the HIV LTR in the absence of other HIV components. As our previous studies indicated that Vpr acts on viral DNA generated in cells during an infection and not on artificially transfected DNA, we infected cells with an integrase-defective MLV vector expressing luciferase from an internal HIV LTR. We found that coinfection with Vpr did not increase the levels of luciferase activity compared to that of coinfection with eGFP. In light of a recent report showing that HIV SIN vectors have promoter activity derived from sequences downstream of the native transcription initiation site (34), we also considered the contribution of additional sequences apart from the core promoter and enhancer sequences from the U3 region of the LTR. However, we found that incorporation of this previously described leader region did not impact the inability of Vpr to increase expression from our HIV LTR-containing MLV vector (data not shown). These studies support the hypothesis that Vpr mediates expression from unintegrated HIV DNA via nontranscriptional mechanisms, as Vpr appears to interact with additional elements that are retained in the integrase-defective SIN HIV vector but are absent in the MLV-based vector.

Vpr increases RNA transcription from HIV IN^–. As indicated above, Vpr appears to be acting at least partially at a transcriptional level on unintegrated HIV DNA, as the loss of the U3 region from the LTR slightly suppresses the extent of Vpr-mediated expression from integration-defective HIV-1-based vectors, i.e., 35-fold from HIV-1 LTR versus 10- to 15-fold from sin18 vector (Fig. 1). Therefore, we examined the effect of Vpr on viral transcription from integrase-defective HIV-1 by quantitative RT-PCR (Table 1). We infected SupT1 cells with VSV-G-pseudotyped HIV, which is capable of expressing all HIV-1 genes except envelope and expresses either functional (Vpr⁺) or truncated Vpr (Vpr^–). We utilized specific primers and probes that would distinguish between unspliced full-length HIV RNA and spliced tat/rev or nef transcripts. Infection of SupT1 cells with integration-competent HIV-1 that contains or lacks Vpr (HIV IN⁺/Vpr⁺ and HIV IN⁺/Vpr^–, respectively) resulted in similar levels of full-length tat/rev and nef viral RNA transcripts. When cells were infected with integration-defective HIV-1, there were detectable levels of all three transcripts examined. The presence of Vpr within integrase-defective virions (HIV D64E/Vpr⁺) resulted in 10- and 8-fold increases in full-length HIV-1 RNA and nef transcripts, respectively, relative to that of Vpr-deleted integrase-defective virus (HIV D64E/Vpr^–). In contrast, there was no effect of Vpr on the levels of tat/rev RNA from integrase-defective HIV. Therefore, Vpr is upregulating transcription from integration-defective HIV, and there is a differential effect on the levels of spliced transcripts.

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TABLE 1. Increased levels of HIV transcripts from integrase-defective HIV by Vpra

Tat is expressed from HIV IN^–, and the levels are not affected by Vpr. Here and in previous studies, the expression of all viral transcripts was demonstrated from integrase-defective viruses in infected cells, including tat/rev transcripts (21, 57, 58). Tat is required for efficient HIV-1 replication and transcription and enhances the LTR-driven expression of integrated virus (27). Previous studies were unable to detect Tat by Western blotting analysis, attributed to the low levels of protein and/or poor reactivity of anti-Tat antibodies, and instead the presence of Tat was inferred by transactivation of LTR-ß-galactosidase constructs. Indeed, we also observed that the level of tat-specific message was 1 to 2 logs lower than the levels of nef and full-length transcripts, respectively. We examined whether Vpr can affect Tat activity from integrase-defective HIV by examining the transactivation of an HIV LTR-luciferase construct introduced by transfection. We transfected HeLa cells with LTR-luciferase, followed by infection with integration-competent or -defective HIV. We first examined the (n-fold) change in transactivation of LTR-luciferase construct upon infection with viruses that do not express Vpr. Infection with integration-competent viruses that do not contain Vpr (HIV IN⁺/Vpr^–) resulted in a 2-log increase of LTR-driven luciferase activity compared to that of noninfected cells (Fig. 2). Similarly, infection with integration-defective viruses (HIV D64E/Vpr^–) also resulted in efficient transactivation. Therefore, there is sufficient Tat produced from integrase-defective HIV to highly transactivate LTR luciferase reporter constructs.

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FIG. 2. Tat is expressed from integrase-defective HIV-1, and the activity is not affected by Vpr. HeLa cells (2 x 105) were transfected with pGL3-LTR luc and plated in 24-well plates. At 24 h posttransfection, cells (4 x 104) were infected at an MOI of 0.5 with 200 ng of p24 antigen of HIV IN+/Vpr+, HIV IN+/Vpr–, HIV D64E/Vpr+, or HIV D64E/Vpr–, as indicated. At day 2 postinfection, cells were harvested and subjected to luciferase analysis. The (n-fold) change in transactivation by Tat is calculated as the level of luciferase activity in the transfected infected cultures relative to that in the transfected uninfected cultures.

We next examined the effect of Tat-mediated transactivation with viruses that express both Tat and Vpr. In our experiments, Vpr does not transactivate HIV plasmid DNA introduced by transfection (40). Therefore, any increase in luciferase activity is due to Tat-mediated transactivation. We found that the presence of Vpr had no effect on Tat-mediated transactivation by either integration-competent or -defective viruses (HIV IN⁺/Vpr⁺ or HIV D64E/Vpr⁺, respectively) because there was a comparable 2-log increase in luciferase, as observed for viruses that do not contain Vpr. Based on these results and the RT-PCR data showing equivalent levels of tat/rev mRNA in the presence or absence of Vpr (Table 1), we also conclude that Vpr does not appear to influence the levels of Tat produced by integrase-defective HIV.

Tat is dispensable for Vpr-mediated transactivation of HIV IN^–. Tat activity is detectable from integrase-defective HIV-1 (Fig. 2), and it has been hypothesized that Tat may be mediating transactivation and expression from unintegrated DNA (19, 56). We investigated the contributions of Tat and Vpr, individually and in combination, to the transactivation of unintegrated DNA derived from integrase-defective and vpr-deleted and tat-deleted HIV-1. To examine the effect of Tat on unintegrated viral DNA expression, we provided Tat in trans to the vpr tat-deleted viruses by infecting HeLa-tat cells, cells that constitutively express Tat. A representative experiment is shown in Fig. 3. In the absence of Vpr, complementation by Tat resulted in a significant increase of expression (24-fold) from integration-competent vpr tat-deleted virus (HIV Vpr^–/Tat^–/IN⁺). In contrast, Tat had a fivefold effect on integration-defective vpr tat-deleted virus (HIV Vpr^–/Tat^–/D64E) (Fig. 3, compare bars 1 and 4). In five independent experiments, Tat alone enhanced expression from integration-competent HIV from 18- to 26-fold, whereas expression from integration-defective HIV increased from 3- to 6-fold. Thus, it appears that Tat, while capable of efficiently transactivating integrated HIV-1, is less potent acting on inducing expression from unintegrated DNA.

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FIG. 3. Tat is dispensable for Vpr-induced expression from integration-defective HIV. HeLa or HeLa-tat cells (2 x 104) were infected at an MOI of 0.1 with vpr-deficient, tat-deficient, luciferase-expressing HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E (25 ng of p24 antigen) and coinfected at an MOI of 1.0 with HR-eGFP or HR-Vpr (200 ng of p24 antigen). Luciferase activity was analyzed at day 2 postinfection. The (n-fold) change in induction in the absence of Vpr and in the presence of Tat was calculated by coinfection with HR-eGFP and HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E in HeLa cells relative to that of HeLa-tat cells. The (n-fold) change in induction in the absence of Tat and in the presence of Vpr was calculated by coinfection of HeLa cells with HR-eGFP and HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E relative to that of the coinfection of HeLa cells with HR-Vpr and HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E. The effect of both Vpr and Tat was calculated by coinfection of HeLa cells with HR-eGFP and HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E relative to that of the coinfection of HeLa-tat cells with HR-Vpr and HIV Vpr–/Tat–/IN+ or HIV Vpr–/Tat–/D64E.

We next determined the effect of Vpr alone on vpr tat-deleted viruses. Coinfection of HeLa cells with integration-defective vpr tat-deleted Vpr^–/Tat^–/D64E virus and a lentiviral vector expressing Vpr, HR-Vpr, induced a 25-fold increase in luciferase expression. In contrast, when Vpr is supplied in trans to the integration-competent vpr tat-deficient Vpr^–/Tat^–/IN⁺ virus, there is only a fivefold effect on expression. In five independent experiments, Vpr alone enhanced expression from tat-deleted integration-defective HIV 12- to 32-fold, whereas expression from integration-competent HIV increased 1- to 5-fold. Thus, Vpr, in the absence of Tat, predominantly increases expression from integrase-defective HIV-1. Conversely, Tat predominantly increases expression from integrated viral DNA.

Both Tat and Vpr have been found to bind to cyclin T1, resulting in synergistic enhancement of transcription from an HIV-1 LTR expression construct (44) We explored the combined effect of Vpr and Tat by coinfecting HeLa-tat cells with the vpr tat-deleted viruses and HR-Vpr. When both Vpr, introduced via coinfection, and Tat, endogenous in HeLa-tat cells, are provided in trans to the integration-competent Vpr^–/Tat^–/IN⁺ virus, there is a 12-fold increase in luciferase expression. This is a significant but lower degree of induction than the 24-fold increase seen with Tat alone (Fig. 3, compare bars 3 and 1). The consistently lower (n-fold) induction of expression from integration-competent HIV by Tat in the presence of Vpr, 8- to 15-fold compared to 18- to 26-fold observed in the absence of Vpr, is likely due to the Vpr-mediated G₂ arrest and the different mechanisms of Tat transactivation in the G₁ and G₂ phases (28). Upon infection with the integrase-defective Vpr^–/Tat^–/D64E virus, there is a 24-fold induction of luciferase activity in the presence of both Tat and Vpr, similar to the 25-fold increase observed with Vpr alone. In five independent experiments, Tat and Vpr together increased expression from integrase-defective HIV 18- to 40-fold, compared to 12- to 32-fold from Vpr alone. Therefore, we did not observe a consistent additive effect of Tat on Vpr-induced expression from unintegrated HIV-1. We conclude that Tat, and not Vpr, is the primary effector of expression from HIV-1 integrated DNA, and conversely, Vpr, and not Tat, is the primary effector of expression from integrated viral DNA.

Vpr increases expression of Nef protein from IN^– HIV. We report above that Vpr upregulates the level of nef transcripts and the luciferase reporter gene is used in place of the nef gene in these experiments. We tested directly whether there is increased expression of Nef protein from integrase-defective HIV in the presence of Vpr. We infected SupT1 cells with integration-competent or -defective HIV, shown in Table 1, that retains a functional nef gene (HIV IN⁺ and HIV D64E, respectively) and analyzed the levels of Nef expression by Western blotting analysis (Fig. 4). Nef was expressed from integration-competent viruses, and these levels were not affected by the absence or presence of Vpr (Fig. 4, compare lane 1 with lane 2). In HIV D64E-infected cells that do not express Vpr, we detected low levels of Nef (Fig. 4, lane 5). In the presence of the functional vpr gene, there was a 15-fold increase in the levels of Nef produced by integrase-defective HIV (Fig. 4, lane 4), protein levels comparable to those seen with integration-competent viruses. Therefore, Vpr stimulates the expression of Nef from integrase-defective HIV.

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FIG. 4. Vpr increases expression of the Nef protein from IN– HIV. SupT1 cells (1 x 105) were infected at an MOI of 0.2 with 200 ng of p24 antigen of wild-type or integrase-defective HIV expressing Vpr (HIV IN+/Vpr+ or HIV D64E/Vpr+; lanes 1 and 4), wild-type or integrase-defective HIV deficient in Vpr (HIV IN+/Vpr– or HIV D64E/Vpr–; lanes 2 and 5), or wild-type or integrase-defective HIV-1 containing Vpr within the virions (HIV IN+/Vpr in trans and HIV D64E/Vpr in trans; lanes 3 and 6) and analyzed 2 days postinfection by Western blotting analysis for the presence of Nef. The arrow indicates the protein band corresponding to Nef. Lysates from cells infected with viruses expressing luciferase in place of nef, IN+/Luc, and D64E/Luc (lanes 7 to 10) were included to indicate the specificity of the anti-Nef antibody.

We also examined whether Vpr, incorporated into virions in the absence of de novo Vpr expression, is sufficient to mediate upregulation of Nef expression from integration-defective HIV-1. This mode of Vpr presentation would mimic the early effects due to release of Vpr from virions during virus uncoating. Vpr was introduced in the same virions as those in the template genome by cotransfecting a Vpr expression construct during virus production, resulting in packaging Vpr within virions that are incapable of de novo synthesis of Vpr. We observed a fivefold increase in Nef expression from integrase-defective HIV that contain Vpr in virions but do not express Vpr from the genome (Fig. 4, lane 6). Thus, virion-associated Vpr alone is sufficient to enhance Nef expression from unintegrated HIV DNA.

Vpr-mediated expression of Nef from HIV IN^– results in CD4 downregulation. Nef has multiple roles in HIV infection and pathogenesis, including downregulation of CD4. We examined whether the increased levels of Nef expressed from integrase-defective HIV in the presence of Vpr can affect CD4 downregulation. SupT1 cells are a T-cell line that expresses CD4 on the cell surface (Fig. 5, 96% CD4-positive population in the mock-infected panel). Infection with integration-competent HIV gave rise to a subpopulation of cells that have lost CD4 expression, and this percentage is not affected by the presence or absence of Vpr (Fig. 5, 24% versus 22% CD4-negative population in HIV IN⁺/Vpr⁺ and HIV IN⁺/Vpr^– virus, respectively). Upon infection with the virus integrase-defective Vpr-containing virus HIV D64E/Vpr⁺, 28% of the cells were CD4 negative, similar to the percentage observed for integration-competent viruses. In contrast with integration-competent virus, the presence of Vpr is required for efficient CD4 downregulation by integrase-defective HIV, as upon infection with integration-defective viruses that do not express Vpr, 7% of SupT1 cells infected with HIV D64E/Vpr^– virus were CD4 negative. This slightly higher number of CD4-negative cells than the 4% present in mock-infected cells may be due to the low but detectable levels of Nef RNA and protein from Vpr-negative integration-defective HIV (Fig. 4 and Table 1).

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FIG. 5. Vpr-mediated expression of Nef from HIV IN– results in CD4 downregulation. SupT1 cells (1 x 105) were infected at an MOI of 0.2 with 200 ng of p24 antigen of wild-type or integrase-defective HIV expressing Vpr (HIV IN+/Vpr+ or HIV D64E/Vpr+), wild-type or integrase-defective HIV deficient in Vpr (HIV IN+/Vpr– or HIV D64E/Vpr–) or wild-type or integrase-defective HIV-1 containing Vpr within the virions (HIV IN+/Vpr in trans or HIV D64E/Vpr in trans), as indicated. Cells were analyzed for CD4 expression at day 2 postinfection by monoclonal antibody staining and flow cytometry. Percentages of the CD4-positive and CD4-negative cells are shown for each sample. The x axis indicates CD4 staining, and the y axis indicates side scatter (SSC).

We also infected cells with integration-defective viruses that contained only Vpr in the virions and did not express Vpr from the genome. The presence of virion-associated Vpr alone in integrase-defective HIV also decreased CD4 expression in infected cells (HIV D64E/Vpr in trans), albeit to a lower extent than that of integrase-defective HIV expressing Vpr (28% versus 17% CD4-negative populations in HIV D64/Vpr⁺ and HIV D64E/Vpr in trans, respectively). This is likely due to the lower levels of Nef expressed from integrase-defective HIV containing Vpr in trans (Fig. 4, compare lanes 4 and 6). We conclude that Vpr packaged in virions, as would be the case in a natural infection, is sufficient for increasing the expression of Nef from integrase-defective HIV and that these levels of Nef are sufficient to downregulate CD4 expression from the cell surface.

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In this study, we examined the potential mechanisms by which Vpr enhances expression from unintegrated HIV DNA. Our genetic analyses indicate that transcription factor binding sites within the HIV-1 LTR are important for Vpr-mediated expression from integration-defective HIV-1-based vectors. Moreover, the presence of Vpr increases the levels of viral transcripts from integrase-defective HIV-1. Therefore, we propose that Vpr acts in part to increase transcription from the HIV-1 LTR in the context of unintegrated viral DNA and that the transactivation is mediated by previously identified cis-acting elements, including the NF-B and Sp1 sites, within the LTR.

However, our studies also indicate that a nontranscriptional component is involved in Vpr's activity with integrase-defective HIV. Vpr is able to enhance expression, albeit at a lower level, from a SIN HIV-based vector deleted in virtually all upstream sequences and most of the known transcriptional elements within the LTR. We found that Vpr did not increase expression from the HIV LTR in the context of an integrase-defective MLV vector, indicating that the HIV LTR alone is not sufficient for Vpr-mediated expression from unintegrated DNA. Last, Vpr increased levels of unspliced gag/pol messages and spliced nef messages but did not affect spliced tat/rev messages. This raises the possibility that Vpr may be functioning via the differential regulation of spliced mRNA. Viral gene expression of HIV is regulated at the level of splicing. HIV currently has five identified 5' splice donor and nine 3' splice acceptor (SA) sites which give rise to over 40 different spliced mRNAs. ASF/SF2, a member of the SR family of proteins that comprises part of the spliceosome, has been implicated in alternative splicing of HIV RNA. Studies have implicated ASF/SF2 in the efficient splicing of rev and nef messages via recognition of the 3' SA5 site and recruitment of U1snRNP to the exonic splicing enhancer element (12). Others have shown competition between ASF/SF2 and hnRNP A1 for an exonic splicing enhancer and an exonic splicing silencer at the 3' SA7 site (35). Depending on the cellular context and the ratio of ASF/SF2 to hnRNP A1, this can result in the activation or repression of A7 utilization. Intriguingly, the HIV 3' SA7 site is retained in the SIN HIV vector but absent in the MLV vector shown in Fig. 1.

The activity of ASF/SF2 is regulated by phosphorylation, possibly in a cell cycle-dependent manner. Dephosphorylated ASF/SF2 stimulates translation in the cytoplasm, while phosphorylated ASF/SF2 stimulates recruitment of SR protein to nascent pre-mRNA transcripts (30, 43). SR proteins are phosphorylated predominantly in the M phase, and studies have shown that Cdc2 kinase, a key regulator of G₂-M transition, can phosphorylate ASF/SF2 in vitro (37), although whether this phosphorylation occurs in vivo and what the significance is of the phosphorylated sites are unclear. Thus, the G₂ arrest induced by Vpr and the altered activity of kinases and phosphatases involved in cell cycle regulation, such as Cdc2 and Wee1 and Cdc25, respectively, may be indirectly affecting HIV alternative splicing through the availability and/or activity of cellular splicing factors, such as ASF/SF2, that act upon HIV-1.

Several lines of study suggest a more direct interaction of Vpr with the splicing machinery. First, Vpr binds to SAP145, a key component of the splicing factor complex SF3B (52). Binding of Vpr to SAP145 results in the exclusion of SAP145 from nuclear speckles and inhibition of the SAP145-SAP49 complex formation. The authors conclude that the interaction of Vpr with SAP145 contributes to the G₂ arrest function of Vpr, as the depletion of SAP145 also leads to cell cycle arrest. More direct evidence for Vpr in splicing regulation has been shown by Kuramitsu et al. (29). They showed that Vpr selectively inhibits pre-mRNA splicing of cellular alpha- and beta-globin-2 genes. Vpr induces accumulation of alpha-globin 2 pre-mRNA containing intron 1 but not intron 2. Preliminary evidence suggests that this is not related to the G₂ arrest phenotype as they observed inhibition of splicing prior to G₂ arrest. We also previously reported that increased expression from unintegrated HIV can still occur when cells are prevented from undergoing Vpr-mediated G₂ arrest by aphidicolin treatment (40). Future studies will focus on the potential link between Vpr and the splicing pathway in specifically upregulating the expression of alternatively spliced HIV transcripts, such as those of nef.

Nef expression from integration-defective HIV-1 has been previously described (21, 57, 58). In quiescent T cells, the early expression of Nef resulted in increased interleukin 2 production and enhanced viral replication (58). Gillim-Ross et al. showed that Nef from integrase-defective HIV downregulated CD4 expression in activated primary lymphocytes (21). Here, we show that Vpr is a critical component in the expression of Nef from integrase-defective HIV. In the presence of Vpr, there is a marked increase in nef transcripts, resulting in functional levels of Nef protein sufficient to decrease CD4 expression. The loss of CD4 from infected cells minimizes the complex formation between CD4 and HIV envelope and increases the production of infectious virus by promoting budding and virion release (7, 31, 42). The downregulation of CD4 soon after infection due to early expression of Nef may prevent superinfection and conserve virions for spread to uninfected cells. In addition to CD4 downregulation, several other activities have been attributed to Nef. Nef protects infected cells from both adaptive and innate cell-mediated immunity by interacting with major histocompatibility complex class I (MHC-I) (16, 45). Nef specifically disrupts HLA A and B expression but not that of HLA C and E, resulting in protection from cytotoxic T lymphocyte (CTL) recognition while maintaining inhibitory signals to NK cell recognition (15). Adnan et al. reported that Nef-mediated MHC-I downregulation affects CTLs recognizing both early (tat, rev, and nef) and late (gag, pol, env, vpr, vpu, and vif) HIV epitopes (2). This suggests that significant Nef-mediated HLA downregulation is occurring prior to the presentation of early protein-derived epitopes. Vpr is found in significant quantities within HIV-1 virions, and here we showed that virion-associated Vpr alone is capable of increasing Nef expression from unintegrated HIV DNA. Thus, the early preintegration expression of Nef, potentiated by virion-associated Vpr, may contribute to increased escape from CTLs targeting epitopes in early proteins.

Numerous reports point to a potential role for unintegrated viral DNA in HIV-1 pathogenesis. The detection of abundant levels of unintegrated DNA in the brain has been associated with the development of AIDS dementia (38, 51). Clinically, high levels of unintegrated DNA correlated with CD4 cell decline and detection of the unintegrated circular HIV-1 intermediates are used as an in vivo marker of continuing HIV-1 replication despite highly active anti-retroviral therapy. By preventing integration, integrase inhibitors can lead to enhanced levels of unintegrated HIV-1 DNA (11, 23). Others have shown that viral gene expression still occurs from integration-competent HIV in the presence of integrase inhibitors (21, 57). These compounds should not affect preintegration events such as viral entry or reverse transcription and are unlikely to affect virion-associated Vpr function. We have shown that virion-associated Vpr protein can induce expression from unintegrated HIV-1. Thus, it is important to study the potential for unintegrated viral DNA to produce viral proteins at levels sufficient to affect physiological conditions as well as the immediate-early function of Vpr that mediates expression from the unintegrated viral DNA.

 
We thank M. Kamata and N. Pariente for critical reading of the manuscript.

This work was supported by National Institutes of Health grants CA70018 and AI43190, by UCLA Center for AIDS Research grant AI28697, and by the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry and Virology Core Facilities.

 
* Corresponding author. Mailing address: UCLA AIDS Institute, Department of Microbiology, Immunology and Molecular Genetics, 11-934 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA 90095-1678. Phone: (310) 825-4793. Fax: (310) 267-1875. E-mail: syuchen{at}mednet.ucla.edu

Published ahead of print on 25 July 2007.

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Journal of Virology, October 2007, p. 10515-10523, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00947-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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