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... Ulrich Schubert,†,‡,1 Samuel A. Williams,*,1 Carlos M. C. de Noronha,* Jason F. Kreisberg,* ...... cutoff Slide-A-Lyzer dialysis cassette (Pierce, Rockford,.
Virology 302, 95–105 (2002) doi:10.1006/viro.2002.1576

HIV-1 Vpr Displays Natural Protein-Transducing Properties: Implications for Viral Pathogenesis Michael P. Sherman,* ,1 Ulrich Schubert,† ,‡ ,1 Samuel A. Williams,* ,1 Carlos M. C. de Noronha,* Jason F. Kreisberg,* Peter Henklein,‡ ,§ and Warner C. Greene* ,¶,2 *Gladstone Institute of Virology and Immunology, San Francisco, California 94141; †Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892; ‡Heinrich-Pette-Institute of Experimental Virology and Immunology, Hamburg, Germany; §Humboldt University, Institute of Biochemistry, 10115 Berlin, Germany; and ¶Departments of Medicine and of Microbiology and Immunology, University of California, San Francisco, California 94143 Received February 25, 2002; returned to author for revision May 3, 2002; accepted May 21, 2002 The 14-kDa Vpr protein of human immunodeficiency virus type 1 (HIV-1) serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest in proliferating T lymphocytes, and the modulation of HIV-1-induced apoptosis. Extracellular Vpr has been detected in the sera and cerebral spinal fluid of HIV-infected patients. However, it is not known whether such forms of Vpr are biologically active. Vpr contains a carboxy-terminal basic amino acid rich segment stretch that is homologous to domains that mediate the energyand receptor-independent cellular uptake of polypeptides by a process termed protein transduction. Similar functional protein-transducing domains are present in HIV-1 Tat, herpes simplex virus-1 DNA-binding protein VP22, and the Drosophila antennapedia homeotic transcription factor. We now demonstrate effective transduction of biologically active, synthetic Vpr (sVpr) as well as the Vpr-␤-galactosidase fusion protein. However, in contrast to other transducing proteins, Vpr transduction is not enhanced by protein denaturation, and Vpr’s carboxy-terminal basic domain alone is not sufficient for its transduction across biological membranes. In contrast, the full-length Vpr protein effectively transduces a broad array of cells, leading to dose-dependent G2 cell-cycle arrest and apoptosis. Addition of Vpr into the extracellular medium also rescues the replication of Vpr-deficient strains of HIV-1 in human macrophage cultures. Native Vpr may thus be optimized for protein transduction, a feature that might enhance and extend the pathological effects of HIV infection. © 2002 Elsevier Science (USA) Key Words: protein transduction; HIV; Vpr; cell cycle.

Re et al., 1995), likely through alterations in the nuclear lamina of cells that lead to dynamic herniations and rupture of the nuclear envelope (de Noronha et al., 2001). Arrest in the G2 phase of the cell cycle provides an intracellular milieu conducive for increased HIV LTR activity and thus viral replication (Goh et al., 1998). Other studies suggest that the prolonged G2 arrest induced by Vpr ultimately leads to apoptosis of the infected cell (Poon et al., 1998; Stewart et al., 1997, 1999, 2000; Yao et al., 1998). Conversely, early antiapoptotic effects of Vpr have also been described which are supplanted later by its proapoptotic effects (Conti et al., 2000). These proapoptotic effects of Vpr may result from direct mitochondrial membrane permeabilization (Ferri et al., 2000), a function that implies the penetration of cellular membranes by Vpr (Piller et al., 1999). Vpr is principally localized in the nucleus despite the lack of any canonical lysine-rich nuclear import signal (Gallay et al., 1996, 1997; Lu et al., 1993). We and others have recently shown that Vpr contains at least two distinct and novel nuclear localization signals (Jenkins et al., 1998; Karni et al., 1998; Sherman et al., 2000). Further, Vpr displays nucleocytoplasmic shuttling properties reflecting the presence of an exportin-1-dependent nuclear export signal (Sherman et al., 2000). The nucleophilic

INTRODUCTION In addition to the genes encoding the structural and enzymatic proteins common to all retroviruses, the human immunodeficiency virus type 1 (HIV-1) genome contains four accessory genes that serve to accelerate viral replication. One of these gene products, viral protein R (Vpr), is a 96 amino acid polypeptide that is packaged into progeny virions through its interaction with the carboxy-terminal p6 Gag domain of the Pr55 Gag precursor protein (Cohen et al., 1990; Paxton et al., 1993; Yuan et al., 1990). HIV-1 Vpr is highly conserved in vivo (Goh et al., 1998; Yedavalli et al., 1998). In the HIV-2 and simian immunodeficiency primate lentiviruses, the function of HIV-1 Vpr is divided between two related proteins, Vpr2 and Vpx (Campbell and Hirsch, 1997; Gibbs and Desrosiers, 1993). Vpr induces G2 cell-cycle arrest in HIV-1infected and/or Vpr-transfected proliferating human cells (Bartz et al., 1996; Goh et al., 1998; He et al., 1995; Hrimech et al., 1999; Jowett et al., 1995; Poon et al., 1998; 1

The first three authors contributed equally to this paper. To whom correspondence and reprint requests should be addressed at Gladstone Institute of Virology and Immunology, P.O. Box 419100, San Francisco, CA 94141-9100. Fax: (415) 826-1817. E-mail: [email protected]. 2

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properties of Vpr are thought to contribute to HIV-1 infection of macrophages (Bukrinsky et al., 1992; Connor et al., 1995; Heinzinger et al., 1994; Miller et al., 1997; Popov et al., 1998). In this regard, although Vpr is not required for HIV-1 infection of nondividing resting T cells (Eckstein et al., 2001), HIV encodes at least three karyophilic proteins including Vpr, matrix p17 MA , and integrase that have been variably proposed to play a role in nuclear uptake of the large viral preintegration complex (PIC, Stokes diameter of 56 nm) (Bouyac-Bertoia et al., 2001; Bukrinsky et al., 1993; Fouchier et al., 1997; Gallay et al., 1996, 1997; Petit et al., 2000; van Schwedler et al., 1994). In addition, a triple-stranded DNA flap formed as an intermediate of reverse transcription may also contribute to nuclear uptake of the viral PIC (Zennou et al., 2000). Structural studies of fragments of Vpr (Luo et al., 1998; Wecker and Roques, 1999) or, more recently, full-length Vpr (Henklein et al., 2000) suggest the presence of a helix-turn-helix between residues 17 and 49 and an amphipathic helix between residues 53 and 78. These helices likely play a key role in homodimerization and the interaction of Vpr with heterologous proteins (Henklein et al., 2000; Zhao et al., 1994a,b). Both helical domains are highly conserved (Sherman et al., 2000) and contribute to the formation of a novel nuclear import signal. The distal helix also contains leucine-rich nuclear export signal whose function is inhibited by leptomycin B (Sherman et al., 2001). The carboxy-terminal region of Vpr contains a basic amino acid rich segment between residues 73 and 96 for which no definite structure has been assigned (Schuler et al., 1999). However, this region is conserved and influences the stability and, potentially, the structure of the entire protein (Yao et al., 1995). This carboxyterminal domain also contains a bipartite, arginine-rich nuclear import signal that can promote nuclear uptake of heterologous proteins through the nuclear pore complex (Jenkins et al., 1998; Sherman et al., 2001). Despite the limiting lipid bilayer comprising the plasma membrane of eukaryotes, an increasing number of proteins have been found to cross this membrane in an apparently energy- and receptor-independent manner. This process—termed protein transduction (Frankel and Pabo, 1988; Green and Loewenstein, 1988) [for reviews, see Hawiger (1999); Porchiantz (2000); Schwarze and Dowdy (2000); Schwarze et al., (2000)]—relies upon the presence of an arginine-rich protein transduction domain (PTD). The carboxy-terminal arginine-rich region of Vpr strongly resembles the PTD present in several transducing proteins, including the HIV transactivating protein Tat, the herpes simplex virus-1 (HSV-1) DNAbinding protein VP22, and the Drosophila antennapedia homeotic transcription factor ANTP (Loret et al., 1991; Vives et al., 1997). While the fusion of these PTDs to heterologous proteins has yielded a novel intracellular delivery system (Anderson et al., 1993; Fawell et al., 1994; Nagahara et al., 1998; Schwarze et al., 1999), it is not

known if any of the native proteins display biologically important protein transduction properties in vivo. Perhaps consistent with this finding, the function of the PTD buried within Tat can be significantly improved by repositioning this domain at the amino-termini of heterologous proteins (Schwarze et al., 1999). Similarly, denaturation of these transducing proteins also enhances their intracellular delivery, likely by more efficiently exposing the PTD (Bonifaci et al., 1995; Ho et al., 2001; Schwarze and Dowdy, 2000). Taken together, these data raise the issue of whether any of the known transducing proteins are truly optimized for protein transduction in their native state in vivo. Vpr has been detected in the sera of HIV-infected patients in quantities that correlate with degree of viremia (Levy et al., 1994). Further, when added extracellularly to the culture medium, both serum-derived and partially purified recombinant Vpr protein preparations activate HIV replication in latently infected T-cell lines, monocyte cultures, and peripheral blood mononuclear cells (PBMCs). These findings have led to the hypothesis that extracellular Vpr can modulate virus replication in vivo, but with no obvious mechanism (Levy et al., 1994, 1995). These studies provided the impetus for our investigation of the ability of extracellular Vpr to cross biological membranes and effect changes in biological responses. Previously we demonstrated that Vpr could enter cells, however, the mechanism and relevance of such entry has not been established (Henklein et al., 2000). Here we demonstrate that Vpr enters cells by protein transduction and can subsequently effect G2 cell-cycle arrest and apoptosis, as well as rescue virus production of Vpr-deficient HIV replicating within infected macrophages when added in trans at nanomolar concentrations. These data thus provide evidence for a biological role for extracellular Vpr and protein transduction. RESULTS Vpr is a transducing protein Protein transduction is defined as rapid cellular uptake of a protein occurring in an energy- and receptor-independent manner (Frankel and Pabo, 1988; Green and Loewenstein, 1988). While we previously showed that sVpr is internalized by cells (Henklein et al., 2000), it was not clear if this process reflected bona fide protein transduction. Therefore, cellular uptake of sVpr was analyzed under conditions of limited energy (4°C). FITC-transferrin, which is internalized by energy-dependent, receptor-mediated endocytosis (Mellman, 1996), was analyzed in parallel as a control similar to previous strategies used to identify other transducing proteins (Derossi et al., 1996). To determine the proportion of each peptide internalized into cells versus that only bound at the cell surface, the cells were treated with trypsin, which di-

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FIG. 1. Full-length sVpr exhibits protein-transducing activity. (A) 293T cells were incubated with sVpr, synthetic fragments of Vpr corresponding to amino acids 1–47 and 52–96, or control proteins transferrin and p6 Gag at 4°C followed by treatment with trypsin (closed bars) or medium (open bars). Under the energy-limiting conditions of low temperature, sVpr was taken up into a trypsin-resistant compartment (internalized), whereas transferrin was not. In contrast to full-length Vpr, neither the 1–47 nor the 52–96 peptide (the latter spanning the entire candidate PTD) mediated effective transduction. (B) 293T cells support transduction of native, but not urea-denatured, FITC-labeled Vpr-␤-galactosidase fusion proteins. M9-FITC protein was used as a negative control in these studies.

gests any trypsin-sensitive putative receptors. Incubation of cells with trypsin after exposure to sVpr or transferrin at 37°C showed little reduction in fluorescence intensity, indicating that both of the proteins had moved off the cell surface into a trypsin-resistant compartment (data not shown). Conversely, when the cells were incubated at 4°C to prevent receptor-mediated endocytosis (Pastan and Willingham, 1981) and exposed to trypsin, transferrin internalization was markedly reduced (indicating successful removal of cell-surface receptors), while entry of sVpr was unaffected (Fig. 1A). These findings indicate that sVpr effectively enters cells under the conditions of limited energy produced at 4°C. The differences observed for Vpr and transferrin internalization suggest that Vpr entry does not involve receptor-mediated endocytosis. Full-length, native Vpr is required for effective transduction The transducing activity of HIV-1 Tat, Drosophila ANTP (Joliot et al., 1998; Perez et al., 1992), and herpes simplex virus VP22 (Elliott and O’Hare, 1997) proteins has been

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mapped to highly basic PTDs. The PTD of Tat corresponds to an 11 amino acid segment that can be fused to heterologous proteins to facilitate their entry into cells (Schwarze et al., 1999). The carboxy-terminal 73–96 amino acid region of Vpr contains an arginine-rich segment closely resembling the PTD present in Tat, VP22, and ANTP. As a first step to mapping the transduction domain within Vpr, we assessed the ability of synthetic fragments of Vpr (amino acids 1–47, 52–96) to recapitulate the transducing activity of the full-length Vpr protein. Neither the control 1–47 amino-terminal portion nor the 52–96 fragment of Vpr containing the arginine-rich segment exhibited detectable transducing activity (Fig. 1A). Both of these Vpr fragments displayed low-level binding to the cell surface that was partially removed by trypsin treatment; however, this binding was no greater than that obtained with the HIV-1 p6 protein and thus likely represents a low level of nonspecific interaction of these proteins with the plasma membrane. Studies with the PTD of Tat fused to heterologous proteins have revealed that protein denaturation improves transduction (Bonifaci et al., 1995; Schwarze et al., 1999). Such denaturation may allow more effective presentation of the PTD to the cell membrane (Derossi et al., 1996). In this regard, transducing proteins may undergo denaturation during their transit through the plasma membrane and then undergo refolding within the cell (Bonifaci et al., 1995; Schwarze et al., 1999). We next assessed whether the efficiency of transduction of a recombinant Vpr-␤-galactosidase fusion protein was similarly enhanced by urea denaturation. This construct was used to show that the transduction properties were not specific to Vpr and because similar experiments were done with a Tat-␤-galactosidase fusion protein (Schwarze et al., 1999). 293T cells were incubated with native or urea-denatured Vpr-␤-galactosidase-FITC, and uptake was then assessed by flow cytometry. To control for nonspecific uptake, cells were incubated with a ␤-galactosidase fusion protein containing the M9 nuclear targeting signal that interacts with intracellular transportin (Jenkins et al., 1998). These studies revealed that the Vpr-␤-galactosidase fusion protein effectively entered cells. However, urea denaturation of the Vpr-␤-galactosidase fusion protein markedly inhibited rather than enhanced such entry (Fig. 1B). These results suggest that the Vpr PTD only functions effectively in the native structure conferred by the presence of the full-length Vpr protein. Taken together, the data in Fig. 1 support the notion that Vpr may be optimized for transduction in its full-length, native state. sVpr transduces multiple cell types rapidly and is biologically active Because of the lack of a requirement for specific receptor engagement, transducing proteins are pre-

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FIG. 2. sVpr rapidly transduces multiple cell types. (A) PBMCs were incubated with Cy3-sVpr (black bars) or Cy3-p6 Gag (white bars) and analyzed for expression of the indicated cell-surface markers. Flow cytometric analysis revealed that CD4 ⫹ and CD8 ⫹ lymphocytes, CD14 ⫹ monocytes, and CD3 ⫺ cell types were all permissive to sVpr transduction. Significant uptake of a Cy3-p6 Gag control protein was not detected in any of these cell types. Error bars represent the mean ⫾ SD of results obtained with cells from four random blood donors. (B) Jurkat T cells were incubated with Cy3-labeled synthetic Vpr or p6 Gag for various times as indicated, and uptake was measured by flow cytometry. sVpr uptake at 5 min occurred as efficiently as at 15 min and beyond.

dicted to enter multiple cell types. When sVpr was incubated with freshly isolated PBMCs, we observed efficient uptake of sVpr, but not of the HIV p6 Gag control, by CD4 ⫹ and CD8 ⫹ lymphocytes, CD14 ⫹ macrophages, and the entirety of the CD3 ⫺ population (Fig. 2A). Additionally, sVpr transduced the vast majority of Jurkat T cells in less than 5 min (Fig. 2B). Together, these data indicate that sVpr transduces a broad array of cells and that this process proceeds with rapid kinetics. When cells were incubated with graded concentrations of fluorescently labeled sVpr, we observed a concentration-dependent increase in cell fluorescence consistent with effective protein transduction (Fig. 3A). However, within the transduced cell population, we detected a dose-dependent change in fluorescence that likely reflects the uptake of different amounts of sVpr (Fig. 3B). We further observed a nuclear pattern of expression of Vpr, a finding that is consistent with the known karyophilic properties of this polypeptide (Henklein et al., 2000). The interaction of Vpr with one or more components of the nucleus may also explain why transduced

sVpr is not significantly transferred between cells under culture conditions (data not shown). Because Vpr induces G2 cell-cycle arrest, we compared the degree of arrest within the transduced population of cells, analyzing the DNA content of cells that had accumulated various amounts of sVpr-Alexa 488. Analysis of subpopulations of sVpr-transduced Jurkat T cells revealed a dose-dependent induction of G2 arrest (Figs. 3B and 3C). Specifically, 37% of the cells containing the highest concentration of sVpr (gate III) displayed a 4N complement of DNA, compared to 33, 15.4, and 11% of cells in gate II, gate I, and within the nontransduced population, respectively. These findings indicate that transduced sVpr induces G2 cell-cycle arrest, underscoring the biological activity of such transduction. Several studies have implicated Vpr in the induction of apoptosis through a caspase-dependent mechanism (Poon et al., 1998; Stewart et al., 1997, 1999, 2000; Yao et al., 1998). Conversely some studies have suggested that Vpr may inhibit programmed cell death (Conti et al., 2000), while other studies have reconciled these differences revealing that Vpr induces early antiapoptotic and late proapoptotic effects (Conti et al., 1998). To evaluate the effects of sVpr protein transduction on apoptosis, Jurkat T cells were incubated with sVpr and levels of apoptosis were assessed by annexin V staining and propidium iodide exclusion (Fig. 4A). Cultures incubated with sVpr exhibited a significantly higher proportion of annexin V staining cells over time than cultures incubated in the absence of sVpr or with the p6 Gag control protein. These results are in agreement with earlier work demonstrating that extracellular Vpr, in the absence of other viral proteins, induces apoptosis (Huang et al., 2000; Piller et al., 1998, 1999). Such effects of sVpr may contribute to the pronounced bystander cell killing observed in HIV-1-infected tissues in vivo (Finkel et al., 1995). Nuclear import of the HIV-1 PIC and infection of nondividing cells similar to macrophages is facilitated by the karyophilic Vpr, p17 matrix, and integrase proteins as well as by a triple-stranded DNA flap formed as an intermediate of reverse transcription (for review, see Sherman and Greene, 2002). Several prior studies have demonstrated that HIV strains lacking Vpr are particularly compromised in their ability to replicate in monocyte-derived macrophages, possibly due to diminished nuclear import of the viral PIC in these nondividing cells. Therefore we examined whether extracellular sVpr could be incorporated into virions during production. Indeed, when NL4-3⌬Vpr was generated from transfected plasmid DNA in the presence of graded amounts of sVpr, we observed a dose-dependent incorporation into pelleted virions (Fig. 4B). To determine whether transduction of a highly purified preparation of Vpr can facilitate HIV replication in macrophages, sVpr (10 nM) was added to monocyte-derived macrophage cultures infected with

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FIG. 3. Transduced sVpr induces G2 cell-cycle arrest in cells in a dose-dependent manner. (A) Jurkat T cells incubated with increasing amounts of Alexa-488-labeled sVpr displayed dose-related increases in fluorescence. FL1-H represents the fluorescence height. (B) A histogram of transduced cells divided into three arbitrary groups according to the (I) low, (II) intermediate, and (III) high level of transduction. (C) Cell-cycle analysis of the three goups of sVpr-transduced cells reveals dose-dependent G2 cell-cycle arrest. Cells with the highest level of transduction displayed the greatest accumulation of 4N DNA content. Lesser though highly significant effects on cell-cycle distribution were observed in cells with intermediate levels of sVpr transduction.

AD8, a macrophage-tropic molecular clone of HIV (Schubert et al., 1995, 1999), or with AD8⌬Vpr, an isogenic virus lacking Vpr. AD8⌬Vpr replicated poorly in macrophages; however, the addition of sVpr to the medium restored infectivity of this viral clone to near wild-type levels (Fig. 5). These results thus both confirm and extend the functional properties of transduced sVpr. DISCUSSION Vpr is a conserved, multifunctional protein that enhances replication of HIV in macrophages and induces G2 cell-cycle arrest in infected proliferating T cells, lead-

ing to enhanced LTR-dependent viral transcription. Circulating Vpr has also been detected in the serum and spinal fluid of HIV-infected patients at a concentration that varies with the degree of p24 antigenemia (Levy et al., 1994). It is, however, unknown whether such extracellular forms of this protein are important for HIV replication or pathogenesis and, if so, how Vpr produces these effects. Our studies now demonstrate that a highly purified, synthetic preparation of Vpr exhibits protein transduction properties, efficiently crossing cell membranes at 4°C in a concentration-dependent but apparently receptor-independent manner. The arginine-rich,

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FIG. 4. Transduced sVpr induces apoptosis and is incorporated into virions. (A) Jurkat T cells were incubated with sVpr (open circles), p6 Gag (filled squares), or medium (filled circles) and assayed for apoptosis by annexin V staining and propidium iodide (PI) exclusion. sVpr-induced apoptosis was significantly increased after 48 h and peaked after 72 h. Cells incubated with medium or p6 Gag did not display similar increases in annexin V staining. This experiment was repeated several times with similar results. (B) Plasmid DNA from NL4-3⌬Vpr was transfected in 293T cells in the presence or absence of graded amounts of sVpr. After removal of cellular debris, viral lysates were ultracentrifuged at 40,000 g and the pellets resuspended in loading buffer prior to PAGE with loading controlled by total virus input as measured by p24 Gag . Wild-type NL4-3 virions and hemaglutinin-Vpr (HA-Vpr) transfected cell lysates were included as positive controls. Note the dose-dependent increase in extracellular sVpr incorporation into virions.

carboxy terminus of Vpr resembles the PTDs present in other well-known transducing proteins. However, in contrast to the PTD present in these other transducing proteins, a fragment of Vpr containing the isolated PTD of Vpr was unable to mediate effective protein transduction. Instead, the full-length Vpr protein was required for transduction. Of note, the Tat PTD functions more efficiently when moved to the amino-terminus of Tat or fused to the termini of heterologous proteins. Further, the PTD sequence can be optimized artificially both by the introduction of specific amino acid substitutions or by denaturation (Schwarze et al., 2000). As such, the protein-transducing properties of the Tat PTD are clearly not optimized for this function in the context of the native protein. The central positioning of this domain may reflect requirements related to its RNA binding, nuclear import, and transcription activities that involve this argi-

nine-rich motif. Nevertheless, full-length extracellular Tat has various biological functions, including the enhancement of interleukin-2 production in activated PBMCs (Ott et al., 1997) and the induction of Kaposi’s sarcoma-like lesions in mice (Ensoli et al., 1994). Indeed, these extracellular functions of Tat have been exploited for vaccine strategies in which Tat or a Tat toxoid serves as an immunogen (Ensoli and Cafaro, 2000; Pauza et al., 2000). In contrast to Tat, it is clear that wild-type Vpr is more active for transduction than fragments containing the putative PTD or full-length, denatured Vpr. Thus, it appears that the carboxy-terminal positioning of the Vpr PTD in the context of the full-length, native Vpr protein generates an effective transducing protein. Moreover, such full-length Vpr proteins are biologically active when transduced as evidenced by the induction of G2 cellcycle arrest and apoptosis and by the enhancement of replication of Vpr-deficient viruses in human monocytederived macrophages. Bystander cell killing is a prominent feature of HIV-1infected tissues (Azad, 2000; Finkel et al., 1995; Xu et al., 1997). Depending on the culture conditions, Vpr has been shown to either increase or decrease apoptosis. Several studies have suggested that recombinant formulations of Vpr can induce cell death in a variety of cell types (Huang et al., 2000; Piller et al., 1998, 1999). It is possible that extracellular Vpr induces apoptosis by the formation of ion-conductive membrane pores (Huang et al., 2000; Piller et al., 1996, 1998, 1999) or even by mitochondrial membrane permeation resulting in disruption of an ion gradient and leading perhaps to release of cytochrome c and activation of the caspase cascade (Ferri et al., 2000). Attempts have been made to use peptides to map the region of Vpr that induces cell death and results suggest that the domain overlaps with an ion-channel-forming phenotype (Piller et al., 1999). However, such mapping experiments employing peptides or partially purified recombinant preparations of Vpr must be viewed with caution because full-length Vpr adopts different conformations depending on solution conditions such as pH and the presence or absence of protein cofactors (Henklein et al., 2000). Our studies now demonstrate that sVpr, in a form that effectively transduces a broad array of target cells and leads to the induction of G2 cell-cycle arrest, can also induce a time-dependent apoptosis when incubated with cultured cells. In one study, exposure to partially purified Vpr induced differential apoptotic effects on unstimulated (proapoptotic) versus CD3-stimulated (antiapoptotic) PBMCs (Ayyavoo et al., 1997). However, this study relied upon CD3 stimulation alone, a situation inherently different from physiologic T-cell receptor (TCR) signaling where coreceptors are also engaged (Chambers and Allison, 1999). It is possible that Vpr influences cells differentially dependent on various states such as TCR activation or even infection (Conti et al., 2000). In fact, it is known that fresh PBMCs from HIV-infected

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FIG. 5. Addition of sVpr to the extracellular medium enhances replication in HIV-1 ⌬Vpr-infected macrophages. Monocyte-derived human macrophages were infected with wild-type (A) or ⌬Vpr (B–D) AD8 macrophage-tropic viruses and were additionally incubated with mock (open circles) or sVpr-containing (filled circles) medium. Viral replication, as measured by reverse transcriptase (RT) activity, was elevated in those cultures complemented with sVpr (10 nM).

patients display a greater propensity for apoptosis upon stimulation in vitro than PBMCs from HIV-seronegative donors (Groux et al., 1992; Meyaard et al., 1992). Such cell killing in vivo might reduce the effectiveness of specific cytotoxic T lymphocytes targeting the virally infected host cells. It will be interesting to determine if serum Vpr contributes to this programmed cell death and whether transducing Vpr participates as an effector in the bystander cell killing observed in HIV infection. HIV strains defective for Vpr cannot replicate efficiently in macrophage cultures. This may reflect a contribution of the nucleophilic Vpr protein within the PIC that facilitates HIV nuclear import in nondividing target cells such as macrophages (Bukrinsky et al., 1992; Connor et al., 1995; Heinzinger et al., 1994; Miller et al., 1997; Popov et al., 1998). However, it is also possible that Vpr alters the state of differentiation in these cells, producing a more favorable host cell environment for viral replication (Subbramanian et al., 1998). This important function of Vpr may explain why this accessory protein is highly conserved in vivo yet rapidly deleted in T-cell cultures (Goh et al., 1998). Our findings show that sVpr enhances viral replication when added in trans to macrophages infected with Vpr-deficient strain of HIV. With the knowledge that Vpr is present in serum and can transduce multiple cell types including monocytes [Fig. 2 and previous work (Henklein et al., 2000)], we now envision a scenario where Vpr may be released locally in tissues after host cell apoptosis. Such circulating forms of Vpr might in turn transduce macrophages targeted for infection and thus facilitate nuclear import of the HIV PIC. In fact, sVpr can

be incorporated into virions when it is present in medium during production of viral stocks in transfected cells (Fig. 4B). Alternatively, since the role of PIC import by Vpr has been questioned (Bouyac-Bertoia et al., 2001), it is possible that Vpr-transduced macrophages may be activated or differentiated in a manner that facilitates subsequent viral infection and replication (Subbramanian et al., 1998). The fact that sVpr is incorporated into virions does not rule out the possibility that Vpr can transactivate replication in a virion-free form. In fact, in monocytederived macrophages infected with wild-type virus infection of MDM there was a slight decrease in viral production in the presence of sVpr, implying a possible balance between transactivation and toxicity. In summary, our findings implicate Vpr as an effective transducing protein. These transducing properties of Vpr may project certain effects of HIV infection to uninfected bystander cells, thus contributing to viral pathogenesis. Indeed, it is intriguing that HIV encodes two proteins, Vpr and Tat, that display transducing properties. These proteins likely act not only intracellularly but also extracellularly in a manner that promotes more efficient growth and spread of HIV-1. Whether disruption of the extracellular function of Vpr would be of therapeutic benefit remains to be explored. MATERIAL AND METHODS Protein synthesis sVpr and relevant fragments were synthesized, purified, and characterized as described (Henklein et al.,

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2000). The 52 amino acid p6 Gag protein comprising the carboxy-terminal mature processing product of the Pr55 Gag precursor derived from the isolate HIV-1 NL-43 (Adachi et al., 1986) was synthesized in an analogous fashion. The purity of peptides was verified by reversephase high-performance liquid chromatography. sVpr was additionally characterized by amino-terminal sequencing with an Applied Biosystems 473A pulsed-liquid-phase sequencer and by positive-ion electrospray ionization mass spectroscopy performed with a fixed Finnigan LCQ ion trap mass spectrometer equipped with an electrospray source as described (Henklein et al., 2000). Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectra were recorded on a Bruker reflex MALDI-TOF mass spectrometer using an N 2 laser (337 nm). sVpr or derived peptides were fluorescently labeled by using a modification of the Alexa 488 labeling kit (A-10235, Molecular Probes) or were end-labeled with a Cy3-like fluorophore (Henklein et al., 2000). Cell culture 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies). Jurkat T cells were cultured in RPMI 1640. Both media were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 ␮g/ml streptomycin. PBMCs from HIV-seronegative random healthy blood donors were obtained from the Stanford Blood Bank (Palo Alto, CA). PBMCs were purified on Ficoll–Paque density gradients (Amersham). sVpr entry assay 293T cells at 80% confluency were preincubated at 4°C for 30 min followed by incubation in DMEM with either 30 ␮g/ml (2.63 ␮M) sVpr-Cy3, 50 ng/ml transferrinpolylysine FITC (Sigma) or medium alone for 30 min at 4 or 37°C. Cells were then washed once with ice-cold phosphate-buffered saline (PBS) and incubated with icecold 0.5% trypsin EDTA or DMEM for 30 min. Trypsin cleavage was halted by adding 3 vol of ice-cold DMEM. Cells were washed once in ice-cold PBS, and fluorescence uptake was measured with a FACScan flow cytometer (Beckton–Dickinson). Because the background fluorescence was altered with temperature and trypsin treatment, the mean fluorescence of the transduced populations (reported as the relative geometric mean of fluorescence) was measured and normalized by subtracting background values obtained in mock-treated control cells. Cell-type independent uptake and cell-cycle assays Jurkat T cells were incubated with sVpr-Alexa 488 (30 ␮g/ml) or control synthetic p6 Gag -Alexa 488 (30 ␮g/ml) in RPMI for 30 min at 37°C. Cells were washed three times

with PBS and analyzed by flow cytometry. Uptake was measured as the percentage of cells displaying greater fluorescence than cells treated with p6 Gag -Alexa 488. PBMCs from four donors were each incubated with sVprAlexa 488 (30 ␮g/ml) or control synthetic p6 Gag -Alexa 488 (30 ␮g/ml) in RPMI for 30 min at 37°C, washed three times with PBS, and stained alone or in combination with CD4-phycoerythrin (PE), CD8-PE, CD3-APC (Beckton– Dickinson), and/or CD14-PE (Pharmingen) fluorescently labeled antibodies. Compensation was employed to ensure proper separation of adjacent fluorescence channels. Cell-cycle analysis was performed on Jurkat T cells after incubation with sVpr-Cy3 for 48 h and fixation in a 2% formaldehyde solution followed by incubation with 1 mg/ml RNaseA and 10 ␮g/ml To-Pro-3 (Molecular Probes) in PBS for 30 min. Cellular DNA content in the fixed cells was then assessed with a FACScan flow cytometer and analyzed with FlowJo Software (Treestar). Urea denaturation Vpr-␤-galactosidase conjugated to FITC and M9-FITC were produced as described (Jenkins et al., 1998) and incubated in 4 M urea with 20 M HEPES for 30 min, followed by a dialysis through a 10,000 molecular weight cutoff Slide-A-Lyzer dialysis cassette (Pierce, Rockford, IL) with PBS for 6 h at room temperature. Apoptosis assay Jurkat T cells were incubated with 30 ␮g/ml sVpr-Alexa 488 for the indicated number of days. Medium containing the indicated peptide was exchanged every 72 h. For analysis, cells were incubated with a 1:50 dilution of antiannexin PE and 1 ␮g/ml propidium iodide (Molecular Probes) in buffer A (calcium/magnesium-free PBS with 1.25 mM CaCl 2, and 2% fetal bovine serum). Cells were washed once with buffer A and analyzed for fluorescence by flow cytometry. Annexin PE-positive, propidium iodide-negative cells were scored as apoptotic. Virus production and monocyte isolation/infection Infection experiments were performed with the HIV-1 chimera NL4-3(AD8) (Freed and Martin, 1994). This chemokine receptor 5-dependent viral strain carries the KpnI-BsmI env determinant of AD8 ⫹ and the V3 loop from a primary monocyte-tropic isolate (Theodore et al., 1996) inserted into pNL4-3 (Adachi et al., 1986). The Vpr-deficient mutant pNL(AD8)-R was generated from pNL43(AD8) by digestion with EcoRI and blunting the resultant “overhangs” with DNA-polymerase I to generate a frameshift mutation in the Vpr open reading frame. Monocytes isolated from blood obtained by leukopheresis from HIV-seronegative donors were elutriated, precultured, and differentiated as described (Ehrenreich et al., 1993; Schubert et al., 1995, 1999). Cells were resuspended in DMEM at a concentration of 0.5 ⫻ 10 6

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cells/ml, and 1.5 ml of cell suspension was replated into 24-well tissue culture plates (Nunc); the cells were allowed to adhere, and then infected with HIV within 3 days. Viral stocks were prepared by transfecting HeLa cells with pNL(AD8)-R or pNL(AD8) plasmids. For transfections, HeLa cells were grown to near confluency in 75cm 2 flasks (5 ⫻ 10 7 cells per flask). Two hours before transfection, the medium was replaced with 15 ml of fresh DMEM, and cells were transfected with lipofectamine 2000 (Life Technologies). Virus-containing supernatants were clarified by centrifugation at 1000 g for 5 min and filtered through a 0.45-␮m filter to remove residual cells and debris. Virions were pelleted by ultracentrifugation in a Beckman SW55 rotor (1 h at 35,000 rpm), resuspended in RPMI1640/fetal bovine serum, and sterilized by filtration. The viral stocks were assayed for reverse transcriptase activity by 32P-TTP incorporation with an oligo(dT)-poly(A) template as described (Willey et al., 1988). Routinely, 10 6 reverse transcriptase units were used to infect 10 6 monocyte-derived macrophages. After 15 h of incubation with virus, the medium was removed and replaced with virus-free medium. The infection status of the cultures was monitored by determining the reverse transcriptase activity in the medium. The cultures were also examined by light microscopy for syncytia formation and scored by counting the number of multinucleated cells per field. ACKNOWLEDGMENTS This work was supported by NIH RO1 AI45234-01A2 to W.C.G. and UCSF-GIVI Center for AIDS Research Grant NIH P30 MH59037. M.P.S. was supported by NIH/NIAID Grant KO8 AI01866. U.S. was supported by grant Schu11/2-1, a Heisenberg grant from the Deutsche Forschungsgemeinschaft, and by a grant from the German Human Genome Research Project, and by NIH/NIDDK RO1 Grant DK59537-01. The authors thank R. Givens and S. Cammack for administrative assistance and J. Carroll and S. Gonzalez for graphics assistance.

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