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Journal of Virology, December 2007, p. 13005-13014, Vol. 81, No. 23
0022-538X/07/$08.00+0     doi:10.1128/JVI.01436-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Association of Nef with p21-Activated Kinase 2 Is Dispensable for Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity in Ex Vivo-Infected Human Lymphoid Tissue{triangledown}

Michael Schindler,1,4 Devi Rajan,1 Anke Specht,1 Carolin Ritter,1 Kati Pulkkinen,2 Kalle Saksela,2,3 and Frank Kirchhoff1*

Institute of Virology, Universitätsklinikum, 89081 Ulm, Germany,1 Institute of Medical Technology, University of Tampere and Tampere University Hospital, 33014 Tampere, Finland,2 Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland,3 Heinrich-Pette-Institute, 20251 Hamburg, Germany4

Received 2 July 2007/ Accepted 5 September 2007


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ABSTRACT
 
Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4+ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.


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INTRODUCTION
 
The accessory Nef protein of human and simian immunodeficiency viruses (HIV and SIV, respectively) is expressed at high levels early during the viral life cycle and is critical for efficient viral replication and persistence in vivo (15, 28, 30). In HIV type 1 (HIV-1)-infected humans and in SIVmac-infected rhesus macaques, high viral loads are associated with the loss of CD4+ T cells and the development of immunodeficiency. Hence, Nef has been characterized as an important virulence factor of primate lentiviruses. A large number of biological effects of Nef that likely explain its importance in vivo have been identified (reviewed in references 4, 67, and 76). These include downmodulation of CD4, CD28, and major histocompatibility complex I (MHC-I), upregulation of the invariant chain (Ii) associated with immature MHC-II complexes, enhancement of virion infectivity, and stimulation of viral replication in primary T cells and ex vivo-infected human lymphoid tissues (HLT) (2, 12, 23, 25, 42, 60, 65, 66, 68, 69, 71). With the exception of nef alleles from HIV-1 and a subset of closely related SIVs, most primate lentiviral Nefs also downmodulate CD3 to suppress T-cell activation and programmed death (62). Thus, while Nef enhances viral pathogenicity in recent or nonnatural hosts, it may protect the natural monkey hosts of SIV against damaging high levels of immune activation.

In addition to modulating various receptors and enhancing virion infectivity, Nef also interferes with cellular signal transduction pathways and interacts with a variety of cellular proteins and kinases (reviewed in reference 55). One of the best-characterized interactions is that with the "Nef-associated" serine/threonine kinase (47, 57). Nef-associated serine/threonine kinase was initially detected as a 62-kDa serine kinase in in vitro kinase assays (IVKAs) (57) and was later identified as p21-activated kinase 2 (PAK2) (5, 53). PAK2 is involved in the regulation of several cellular processes, e.g., cytoskeleton rearrangement, cell morphology, motility, apoptosis, and gene transcription, and is activated in response to a variety of cellular stresses (reviewed in references 6, 13, and 14). Usually, endogenous PAK2 is activated by binding of the GTP-bound form of p21 GTPase Rac1 or Cdc42, which triggers a cascade of autophosphorylation events (80). The mechanism by which Nef activates PAK2 is poorly understood. Nef is thought to activate PAK2 through a multiprotein complex, but it has proven difficult to identify its components (5, 33, 51). The interaction of Nef with PAK2 is conserved between different groups of primate lentiviruses (32, 34, 58), suggesting a relevant biological role. Notably, however, it has been shown that the majority of Nef alleles interact with activated PAK2 but fail to or only poorly activate it itself (51).

It has been proposed that the interaction of Nef with PAK2 might play an important role in T-cell activation (36) and hence in stimulating virus replication in HIV-1-infected cells (3, 64, 75, 81). Moreover, it has been implicated in inducing Fas-Fas ligand expression (82) and as an effector of Nef in inhibiting Bad-mediated apoptosis (79), although the latter effect could not be confirmed in a subsequent study (61). Currently, the biological significance of the interaction of Nef with PAK2 is still poorly understood. The major obstacle for conclusive studies was that most mutations in Nef disrupting this interaction, i.e., those located in the N-terminal myristoylation signal, in the proline-rich region or "PXXP" domain, or in arginine residues R105 and R106, have pleiotropic effects on Nef function (22, 40, 48, 78). For example, the PXXP motif of HIV-1 Nef also mediates the interaction with the SH3 domains of the Src tyrosine family kinases and Vav, as well as effects of Nef on cellular calcium metabolism (41), and is involved in efficient MHC-I downmodulation (19, 39, 55). Residues R104 and R105 are critical for PAK2 binding (29, 57) but most likely also for the correct Nef core structure, and changes in these residues have deleterious effects on multiple Nef functions. More recently, however, a hydrophobic binding surface involving residues 85, 89, 187, 188, and 191 in Nef has been identified as important for PAK2 association but not for downregulation of CD4 or MHC-I (1, 22, 48).

In particular, mutations in F191 were proposed to impair or disrupt Nef association with PAK2 without affecting other Nef functions (1, 22). However, only Nef-mediated modulation of CD4 and MHC-I was analyzed in these studies. To further assess whether mutations in F191 indeed selectively affect Nef's association with PAK2 and hence to allow us to elucidate the biological significance of this interaction, we performed a comprehensive analysis of HIV-1 Nef mutants containing specific changes to this hydrophobic residue. In agreement with previous reports (1, 22), we found that an F191H mutation impaired and an F191R mutation disrupted the association of Nef with PAK2. Unexpectedly, our results demonstrated that these changes also impair Nef-mediated enhancement of virion infectivity in P4-CCR5 cells but not in another indicator cell line, TZM-bl, or in human peripheral blood mononuclear cells (PBMCs). In comparison, they had little, if any, effect on downmodulation of CD4 and MHC-I, upregulation of Ii associated with immature MHC-II complexes, the levels of apoptosis in HIV-1-infected PBMC cultures, induction of NFAT activation, or the ability to stimulate HIV-1 replication in primary T cells or ex vivo-infected HLT. In conclusion, our results suggest that the interaction of Nef with PAK2 does not play a major role in T-cell activation and HIV-1 replication or CD4+ T-cell depletion in HLT.


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MATERIALS AND METHODS
 
Proviral constructs. HIV-1 NL4-3 proviral constructs carrying the intact NA7 or Pex nef allele or a disrupted nef gene followed by an internal ribosome entry site (IRES) and the enhanced green fluorescent protein (EGFP) gene have been described previously (60, 61). Briefly, the nef-defective control constructs contained a premature stop codon at position 40 of the HIV-1 NL4-3 nef gene (nef*) or the same stop codon combined with a second stop codon at position 3 and a mutation of the initiation codon (nef–). Splice overlap extension PCR was used to introduce mutations F191H and F191R into the NA7 and Pex nef alleles. HIV-1 NL4-3 nef mutants without the IRES-EGFP element were constructed by cloning env-nef fragments derived from the NL4-3-based Nef-IRES-EGFP constructs (60, 61) into HIV-1 NL4-3nef+{Delta}1{Delta}2 (43), using the single HpaI and MluI restriction sites in env and just downstream of the nef gene, respectively. The integrity of all PCR-derived inserts was verified by sequence analysis.

Cell culture and virus stocks. P4-CCR5 and 293T cells were cultured as described previously (16, 44, 46). P4-CCR5 (11, 20) and 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. The human monocytic THP-1 cell line (74) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics. PBMCs from healthy human donors were isolated using lymphocyte separation medium (Biocoll separating solution; Biochrom) and either stimulated for 3 days with phytohemagglutinin (PHA; 1 µg/ml) and cultured in RPMI 1640 medium with 10% FCS and 10 ng/ml interleukin-2 (IL-2) prior to infection or infected with HIV-1 immediately after isolation, cultured for 3 days in RPMI 1640 medium (supplemented with 10% FCS and 10 ng/ml IL-2), and subsequently PHA activated (1 µg/ml) for 3 days. To generate viral stocks, 293T cells were either transfected with the proviral NL4-3 constructs with an IRES-EGFP element alone or cotransfected with the HIV-1 Nef/EGFP constructs and a plasmid (pHIT-G) expressing the vesicular stomatitis virus G protein (60, 61). The latter was used to achieve high initial infection levels for functional analysis. Virus stocks were quantified using a p24 antigen capture assay provided by the NIH AIDS Research and Reference Reagent Program and were stored at –70°C.

Infectivity assays. Virus infectivity was determined using P4-CCR5 cells as described previously (44, 46). Briefly, the cells were sown into 96-well dishes in a volume of 100 µl and infected after overnight incubation with virus stocks containing 1 to 5 ng of p24 antigen. At 3 days postinfection, viral infectivity was detected using a Gal screen kit from TROPIX as recommended by the manufacturer. ß-Galactosidase activities were detected in relative light units per second, using a Berthold microplate luminometer.

IVKAs. IVKAs were performed as described previously (53, 54). Briefly, 293T HEK cells were cotransfected with pEBB-PAK2-MycHis-HA, pEBG-Cdc42V12, and pEBB-lacZ (54) and with expression vectors for the different AU1-tagged nef alleles by using the Lipofectamine transfection agent (Gibco BRL) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline and lysed in IVKA lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin/ml). Lysates were cleared and corrected for ß-galactosidase activity by an o-nitrophenyl-ß-D-galactopyranoside assay. The corrected lysates were used for Western blot analysis with anti-Myc (for PAK2; Sigma) and anti-AU-1 (for Nef and Vpu; Covance). Immunoprecipitation was performed with anti-AU1-coupled protein G-Sepharose beads. After washing of the immunoprecipitates three times with IVKA lysis buffer and twice with IVKA buffer (50 mM HEPES [pH 7.4], 5 mM MgCl2), the beads were subjected to an IVKA for 30 min at 30°C. Proteins were separated in sodium dodecyl sulfate (SDS)-8% polyacrylamide gel electrophoresis (PAGE) gels for IVKAs and in 12% gels for Western blot analysis.

Flow cytometric analysis. CD4, TCR-CD3, MHC-I, CD28, CXCR4, and EGFP reporter expression in human PBMCs and Ii expression in THP-1 cells transduced with HIV-1 (NL4-3) constructs coexpressing Nef and EGFP were measured as described previously (60, 62, 63). IL-2 receptor (IL-2R) expression was measured by standard fluorescence-activated cell sorter staining, using a CD25 monoclonal antibody (MAb) (clone M-A251; BD Pharmingen).

NFAT induction. Jurkat cells stably transfected with an NFAT-dependent reporter gene vector (21) were either left uninfected or transduced with HIV-1 Nef/EGFP constructs expressing various nef alleles. Except for cells used as controls, cultures were treated with PHA (1 µg/ml; Murex). Luciferase activity was measured, and n-fold induction was determined by calculating the ratio of measured relative light units of treated samples to that of untreated samples as described previously (21).

Induction of PBMC activation, apoptosis, and viral replication. Human PBMCs were first stimulated with PHA (1 µg/ml) for 3 days. Subsequently, the cells were cultured in RPMI 1640 (with 10% FCS and 10 ng/ml IL-2), infected with various HIV-1 EGFP/Nef constructs, and cultured for another 2 days as described previously (62). At this time, the PBMCs expressed very low levels of IL-2R and hence had a resting phenotype. Thereafter, the PBMCs were treated a second time with PHA, and IL-2R expression levels were measured by fluorescence-activated cell sorter analysis 3 days later. The frequency of virally infected apoptotic cells was determined using an annexin V apoptosis detection kit (BD Bioscience) as recommended by the manufacturer. To assess the ability of Nef to promote viral spread and infectivity, PHA-stimulated PBMCs were infected with HIV-1 stocks containing 1 ng p24, and the percentage of virally infected GFP+ cells was determined at 2- or 3-day intervals.

Ex vivo-infected HLT. HIV-1 replication and cytopathicity in ex vivo-infected HLT were determined as described previously (24, 26, 56). Briefly, human tonsillar tissue removed during routine tonsillectomy was received within 5 h of excision. The tonsils were washed thoroughly with medium containing antibiotics and sectioned into 2- to 3-mm3 blocks. These tissue blocks were placed on top of collagen sponge gels and infected with virus stocks containing 0.5 ng p24 antigen essentially as described previously (24, 26, 56). Supernatants were collected at 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. Flow cytometry was performed on cells mechanically isolated from control and infected tissue blocks, and depletion of CD4+ T cells was quantified as described previously (24, 26, 56). For determination of the CD4+/CD8+-T-cell ratio, cells were stained for surface markers by using anti-CD3-fluorescein isothiocyanate, anti-CD4-allophycocyanin, and anti-CD8-tricolor.


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RESULTS
 
Mutations in F191 affect the association of Nef with PAK2. It has been proposed that alterations in F191 affect PAK2 association but not other Nef functions (1, 22, 48). To further assess whether changes in this hydrophobic residue may allow a meaningful analysis of the biological relevance of Nef-PAK2 binding, we introduced mutations F191H and F191R into the NA7 and Pex Nef alleles. NA7 Nef is a highly active natural HIV-1 nef allele that has been characterized in previous studies (27, 60). The Pex consensus nef allele was generated based on the analysis of nef sequences derived from 91 HIV-1-infected individuals at different stages of disease (10, 31). To examine the effect of the F191H/R mutations on Nef expression levels and PAK2 binding, we cotransfected 293T cells with constructs expressing the various nef alleles, a myc-tagged form of PAK2, and the dominant-active p21 GTPase Cdc42V12. F191H/R mutations had no detectable effect on Nef expression levels (Fig. 1). The wild-type NA7 and Pex Nef proteins interacted efficiently with PAK2 activity (Fig. 1, lanes 3 and 6). In the context of both nef alleles, the F191H mutation significantly reduced and the F191R mutation entirely abolished Nef-associated PAK2 autophosphorylation activity (Fig. 1, lanes 4, 5, 7, and 8). These results suggested that the F191H/R Nef mutants may be useful for studying the biological significance of PAK2 interaction.


Figure 1
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  		FIG. 1. Mutations in amino acid residue 191 affect the interaction of HIV-1 Nef proteins with PAK2. 293T cells were cotransfected with expression plasmids for myc epitope-tagged PAK2, Cdc42V12, and the indicated AU1-tagged Nef or Vpu proteins. An aliquot of cell lysate was directly separated by SDS-PAGE and analyzed for PAK2 (upper panel) or Nef and Vpu (middle panel) expression by Western blot analysis using myc- and AU-1-specific antibodies, respectively. To detect PAK2 interaction, Nef immunocomplexes were precipitated with anti-AU1 antibody, subjected to IVKA, and separated by SDS-PAGE (lower panel). Control, cells were cotransfected only with myc epitope-tagged PAK2 and Cdc42V12.

Effect of F191H/R changes on Nef-mediated receptor modulation. To study the effect of the F191H/R mutations on Nef function in virally infected primary human cells, we cloned all nef alleles into a replication-competent HIV-1 NL4-3-based proviral vector constructed to coexpress Nef and EGFP at correlating levels from a bicistronic RNA (60-62). Flow cytometric analysis of PBMCs transduced with the viral constructs showed that the F191H and F191R changes did not disrupt the ability of Nef to downmodulate cell surface expression of CD4, MHC-I, and CD28 (Fig. 2A). However, the F191R change reduced the ability of Nef to downmodulate CD4 and MHC-I in the context of the NA7 but not the Pex nef allele (Fig. 2B). Moreover, both mutations affected the efficiency of CD28 downregulation. In agreement with published data (62), none of the HIV-1 nef alleles downmodulated CD3. Moreover, all HIV-1 Nef alleles investigated only weakly affected CXCR4 expression by virally infected PBMCs (Fig. 2). It was established previously that Nef efficiently upregulates Ii, presumably to impair MHC-II antigen presentation (60, 69). We utilized the human monocytic leukemia THP-1 cell line to study Ii upmodulation because it shares many properties with human monocyte-derived macrophages (74) and coexpresses high levels of both MHC-I and MHC-II. As expected (63), THP-1 cells infected with HIV-1 constructs expressing the NA7 and Pex nef alleles showed drastically enhanced levels of Ii surface expression (Fig. 2). The F191H and F191R changes did not reduce the potency of Nef-mediated Ii upregulation. Altogether, our data show that the NA7 and Pex F191R and F191H mutant Nefs are capable of modulating various cellular receptors, albeit with reduced efficiency in some cases.


Figure 2
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  		FIG. 2. Effect of F191H/R mutations on Nef-mediated receptor modulation. (A) PBMCs or THP-1 cells were either mock transduced or transduced with vesicular stomatitis virus glycoprotein-pseudotyped NL4-3-based reporter viruses expressing GFP alone (Nef–) or together with the indicated nef alleles and were analyzed by flow cytometric analysis. Values give mean fluorescence intensities for the range of EGFP expression used to calculate the modulation of the various receptors. Similar results were obtained in an independent experiment. (B) Relative CD4, MHC-I, CD28, CXCR4, CD3, and Ii cell surface expression levels on PBMCs or THP-1 cells (Ii) infected with the indicated HIV-1 Nef/EGFP constructs. Receptor expression levels on cells infected with the nef– control construct were set to 100%, and values are averages ± standard deviations (SD) from two experiments.

Impact of Nef-PAK2 association on T-cell activation and apoptosis. It has been proposed that the interaction of Nef with PAK2 may alter T-cell activation (36). To evaluate whether PBMCs infected with HIV-1 variants that do or do not interact with PAK2 show phenotypic differences in their activation status, we measured expression of IL-2R, a late T-cell activation marker. As shown in Fig. 3A, PBMCs infected with nef– HIV-1 NL4-3 expressed slightly higher levels of IL-2R than did uninfected control cells. For comparison, the levels of IL-2R expression remained unaltered in cells infected with the wild-type NA7 and Pex Nef HIV-1 variants but were slightly enhanced in PBMCs infected with the F191R and F191H HIV-1 Nef mutants (Fig. 3A). This minor difference could be due to the reduced ability of the F191R/H Nefs to downmodulate CD28 (Fig. 2). It has been suggested that PAK2 is an effector of Nef in inhibiting Bad-mediated apoptosis (79). However, we could not confirm an antiapoptotic effect of endogenous HIV-1 Nef (61). In agreement with our previous results, PBMCs infected with HIV-1 variants expressing either no Nef or Nef alleles differing in their ability to interact with PAK2 showed only marginal differences in the levels of infection-associated apoptosis (Fig. 3B). Thus, the association of Nef with PAK2 or a lack thereof did not have a marked effect on the level of IL-2R expression or apoptotic death in HIV-1-infected primary human cells.


Figure 3
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  		FIG. 3. Levels of IL-2R expression and apoptosis in PBMCs infected with HIV-1 Nef variants differing in PAK2 interaction. Analysis of human PBMCs was done following transduction with the indicated HIV-1 Nef/EGFP constructs. IL-2R levels (shown relative to those on cells infected with the nef– control construct) (A) and apoptosis (B) were determined at 3 days poststimulation as described in Materials and Methods. The values shown are average values ± SD derived from two experiments.

To further examine whether the six HIV-1 nef alleles may differ in their ability to modulate the responsive of T cells to activation, we transduced Jurkat T cells containing an NFAT-dependent luciferase reporter gene (21) with the HIV-1 IRES-EGFP Nef mutants. NFAT is an important regulator of IL-2R gene expression, one of the hallmarks of T-cell activation. Compared to uninfected Jurkat T-cell cultures, those infected with the nef– and nef* HIV-1 constructs showed an average 3.5-fold increase of NFAT activity after stimulation with PHA (Fig. 4). As expected from previous studies (21, 62), this increase in NFAT activation was three- to sixfold stronger in cells infected with HIV-1 constructs expressing the NA7 and Pex nef alleles. The NA7 Nef variants were usually more active than the Pex Nefs in this assay (Fig. 4). On average, the levels of NFAT-dependent luciferase activity measured in cells infected with the HIV-1 Nef mutants were even slightly higher than those detected in cultures infected with the wild-type NA7 and Pex HIV-1 Nef constructs (Fig. 4). Thus, the F191R and F191H mutations did not impair the ability of Nef to enhance the responsiveness of HIV-1-infected T cells to activation.


Figure 4
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  		FIG. 4. Induction of NFAT by Nef proteins showing differential interaction with PAK2. Jurkat cells stably transfected with an NFAT-dependent luciferase reporter gene were either left uninfected (mock) or transduced with the indicated HIV-1 Nef/EGFP variants. The levels of luciferase activity were determined at 16 h poststimulation. The values shown are averages ± SD derived from sextuplet transductions. RLU, relative light units.

Effect of F191R/H mutations on Nef-mediated enhancement of viral infectivity and replication. Enhancement of virion infectivity is a well-established Nef function (12, 42, 65), and it has been suggested that HIV-1 Nef interaction with PAK2 correlates with enhanced virion infectivity in vitro, although these results are difficult to interpret because the mutations used had pleiotropic effects (78). We first infected P4-CCR5 and TZM-bl indicator cells with virus stocks containing normalized quantities of p24 antigen derived from 293T cells transiently transfected with the different proviral constructs. We found that the wild-type NA7 and Pex Nef proteins enhanced virion infectivity 16.9- and 31.3-fold, respectively, in P4-CCR5 cells (Fig. 5A) and 3.2-and 4.8-fold, respectively, in TZM-bl cells (Fig. 5B). Remarkably, the F191R mutation almost entirely disrupted the ability of both nef alleles to promote virion infectivity in P4-CCR5 cells, whereas the HIV-1 variants containing the F191H substitution showed a phenotype intermediate between those of nef-defective and wild-type HIV-1 (Fig. 5A). In contrast, the F191R/H mutations had no significant disruptive effects on HIV-1 infection of TZM-bl cells (Fig. 5B). This result was confirmed at a 10-fold lower viral dose and was therefore not due to overinfection of the TZM-bl cells (data not shown). To examine the impact of Nef on HIV-1 infectivity in primary cells, we infected PBMCs from different donors with virus stocks containing normalized quantities of p24 and determined the numbers of infected GFP+ cells 3 days later. We found that the F191R/H mutations in Nef did not significantly impair the efficiency of HIV-1 infection of human PBMCs (Fig. 5C). Altogether, our data showed that Nef expression in the 293T producer cells enhanced HIV-1 infectivity for all cell types analyzed, although the effects were most pronounced in P4-CCR5 cells. The F191R change disrupted and the F191H mutation impaired the ability of Nef to enhance virion infectivity in P4-CCR5 cells but had no disruptive effects in TZM-bl cells or PBMCs (summarized in Fig. 5D). Notably, these cell type-dependent differences were not biased by variations in the viral stocks because P4-CCR5 and TZM-BL cells as well as PBMCs were all infected in the same experiment, using aliquots of the same virus stocks. Moreover, the differences in the magnitudes of the Nef effects were confirmed at different viral doses (data not shown).


Figure 5
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  		FIG. 5. Effects of mutations in residue F191 on Nef-mediated enhancement of virion infectivity. P4-CCR5 (A) and TZM-bl (B) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (C) PBMCs from two donors were infected in triplicate with the indicated HIV-1 IRES-EGFP constructs (1 ng p24 antigen), and the number of GFP+ cells was quantified 3 days later. The values shown are average values ± SD. (D) Effects of F191R/H mutations on infectivity enhancement by Nef in different target cells. All infectivities are given as percentages of the corresponding parental NA7 or Pex Nef allele infectivity. The numbers above the bars indicate average values.

It has been shown that the ability of Nef to promote virion infectivity does not correlate with its ability to enhance viral replication in primary PBMC cultures (37), and hence both represent independent Nef functions. To assess viral spread, we infected PBMCs with HIV-1 IRES-EGFP constructs expressing the wild-type X4-tropic Env protein and either no Nef or the various NA7 and Pex nef alleles and determined the numbers of virally infected GFP+ cells at different time points. We found that all six HIV-1 recombinants containing intact nef alleles spread with higher efficiency than the two nef-defective control constructs in both prestimulated PBMC cultures (Fig. 6A) and PBMCs that were infected immediately after isolation and PHA activated 3 days later (Fig. 6B). The F191R/H mutations in Nef, however, did not significantly impair the spread of HIV-1 in prestimulated or "resting" PBMC cultures.


Figure 6
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  		FIG. 6. Spread of HIV-1 NL4-3 F191R/H variants in PBMC cultures. PBMCs were either stimulated with PHA (1 µg/ml) for 3 days and subsequently infected with virus stocks containing 0.5 ng p24 antigen (A) or infected with virus stocks containing 1.0 ng p24 antigen immediately after isolation and treated with PHA 3 days later (B). The number of HIV-1-infected GFP+ cells was determined by flow cytometric analysis at the indicated time points. All curves represent average values obtained from duplicate infections. The results were confirmed using PBMCs from different donors.

Association of Nef with PAK2 is dispensable for efficient HIV-1 replication in ex vivo-infected HLT. It has been shown that Nef greatly enhances HIV-1 replication in ex vivo-infected HLT (25). This experimental system is likely to be relevant for the pathogenesis of AIDS because HLT is one site where the bulk of virus replication and the key pathogenic events occur in vivo (24). Moreover, ex vivo-infected HLT does not require exogenous stimulation to allow efficient replication of HIV-1 and should therefore allow assessment of whether the interaction of Nef with PAK2 may promote HIV-1 replication by enhancing T-cell activation. As expected from published data (25), all HIV-1 NA7 and Pex Nef variants replicated with faster kinetics and substantially higher efficiencies than those of the nef– control virus (an example is shown in Fig. 7A). On average, intact NA7 and Pex nef genes increased virus production between four- and sixfold (Fig. 7B). The F191H/R mutations did not significantly impair the efficiency of virus replication (Fig. 7A) and had no significant effect on p24 production in ex vivo-infected HLT (Fig. 7B). Moreover, we found that all six HIV-1 NA7 and Pex Nef variants depleted about 60% of CD4+ T cells from the tissue blocks by the end of the 15-day culture period, whereas only about 20% of CD4+ T cells were depleted in nef-defective HIV-1 infection (Fig. 7C). Thus, mutations in Nef that disrupt its interaction with PAK2 and virion infectivity in vitro did not significantly reduce its ability to enhance HIV-1 replication and cytopathicity in ex vivo-infected HLT.


Figure 7
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  		FIG. 7. Replication and cytopathicity of HIV-1 NL4-3 F191R/H variants in HLT ex vivo. (A) Representative replication kinetics of wild-type NL4-3 and the indicated nef recombinants. (B) Average virus production in HLT infected ex vivo. (C) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from four donors were infected with the indicated nef variants, and cumulative p24 production by the tissue blocks over 15 days (B) or CD4+ T-cell depletion at the end of culture (C) was determined as described in Materials and Methods. p24 production is given as a percentage compared to cultures infected with the virus expressing NA7 Nef (100%). The values shown are means ± standard errors of the means.


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DISCUSSION
 
In the present study, we show that the amino acid changes F191R and F191H in the NA7 and Pex Nefs affect the association of Nef with PAK2 and profoundly compromise the enhancement of virion infectivity in P4-CCR5 cells. In contrast, these mutations have little or no effect on Nef-mediated modulation of CD4, MHC-I, and Ii surface expression, induction of NFAT, and enhancement of HIV-1 infection in TZM-bl cells and PBMCs. These results expand those of previous reports (1, 22, 48) proposing that F191 is part of a hydrophobic binding surface specifically involved in PAK2 association but not in other Nef functions. Most importantly, disruption of the association of Nef with PAK2 did not impair HIV-1 replication and cytopathicity in PBMCs and ex vivo-infected HLT. Since PAK2 association is a conserved property of HIV-1, HIV-2, and SIV Nef proteins (29, 32, 34), it most likely provides some selective advantage for these viruses in vivo in infected human and simian hosts. Nonetheless, our current knowledge clearly suggests that other Nef functions are more important in vivo, i.e., downmodulation of CD4 and MHC-I and stimulation of viral replication were all shown to contribute to efficient viral persistence and the pathogenesis of AIDS in the SIVmac-macaque model (7, 17, 45, 70). In contrast, the selective pressure for PAK2 binding in vivo is weak, and restoration of this interaction is not required for the development of high viral loads and fatal disease in SIVmac-infected rhesus macaques (9, 29, 34, 57).

Conflicting results have been reported on the relevance of PAK2 association for infectivity enhancement by Nef (38, 78). PAK2 is involved in cytoskeleton rearrangements (6, 14, 19) and could potentially affect virion infectivity by facilitating the penetration of the actin cytoskeleton by the viral preintegration complex (8). We found that the F191H and F191R changes had comparable disruptive effects on the ability of the NA7 and Pex Nef alleles to interact with PAK2 and to enhance HIV-1 infectivity in P4-CCR5 indicator cells. At first view, our data support a role for Nef's ability to bind PAK2 in infectivity enhancement. However, the disruptive effects of the F191R/H mutations were cell line specific and were not observed in TZM-bl cells and PBMCs. Moreover, we previously found that the HIV-2 CBL Nef, which effectively binds and activates PAK2 (51), only weakly enhances virion infectivity in P4-CCR5 cells (46). Similarly, we found that the ability of primary HIV-1/SIVcpz nef alleles to interact with PAK2 does not correlate with their activity in promoting virus infectivity (32; data not shown). Thus, Nef-mediated infectivity enhancement and PAK2 association are not functionally linked.

It has been established that Nef expression in the virus-producing cell enhances virion infectivity at an early step of the viral replication cycle (12, 42, 65) and might require dynamin 2 (49). However, it is largely unclear which Nef-dependent modifications of the HIV-1 particles account for its effect on infectivity. Enhancement of cytoplasmic delivery by increased CD4- and chemokine receptor-dependent HIV-1 entry (59), perhaps due to enhanced cholesterol content of progeny virions (83), reduced susceptibility of virions to proteasomal degradation in the target cells (52), and facilitated transport of the viral genome through the cortical actin network (8) were all proposed to play a role in Nef-mediated HIV-1 infectivity enhancement. We found that expression of NA7 and Pex Nef alleles enhanced HIV-1 infectivity about 20- to 50-fold in P4-CCR5 indicator cells but only up to 5-fold in TZM-bl cells and PBMCs. As noted above, the disruptive effect of the F191R change on infectivity enhancement by Nef was observed only in P4-CCR5 cells. Both P4-CCR5 and TZM-bl indicator cells were originally derived from the HeLa cell line (11, 16, 20, 50, 77). An important difference is that TZM-bl cells are more susceptible to HIV-1 infection and express about 15-fold higher levels of CD4 but 5-fold lower levels of CXCR4 than do P4-CCR5 cells (data not shown). Our observations that the levels of CD4 expression and the magnitudes of the Nef effects on infectivity were similar in TZM-bl cells and PBMCs further support the hypothesis that Nef is less critical for HIV-1 infection of target cells that are highly susceptible to infection and express high levels of CD4 (73). In agreement with this possibility, the potency of CD4 downmodulation, not that of infectivity enhancement by Nef, correlates with the efficiency of viral replication in primary lymphocyte culture and ex vivo-infected HLT (26, 37). Apparently, at least three HIV-1 Nef activities, i.e., CD4 downmodulation, activation of resting T cells, and enhancement of virion infection, contribute to efficient viral replication. Obviously, the relative importance of these Nef functions may vary depending on the activation status and CD4 expression level of the target cells. Results obtained in the SIVmac-macaque model and derived from long-term survivors of HIV-1 infection clearly suggest that CD4 downmodulation and alteration of T-cell activation by Nef are important for efficient viral spread in vivo and the pathogenesis of AIDS (7, 17, 27, 72). In contrast, the relevance of Nef-mediated infectivity enhancement in vivo is less clear but is supported by the observation that this function is conserved between HIV-1 and HIV-2 nef alleles (12, 46) and apparently contributes to efficient spread of SIVmac in infected rhesus macaques (7). However, the usage of HeLa-derived cell indicator lines is a caveat of most studies on Nef-mediated infectivity enhancement. To avoid possible artifacts, it will be important to define the effects of Nef on HIV-1 infectivity in producer and target cells that are relevant for viral spread in vivo, i.e., CD4+ T cells and macrophages.

There has been a lot of speculation about a possible role of Nef-PAK2 association in the activation of resting T cells and hence the stimulation of HIV-1 replication (1, 18, 22, 35, 36, 48). The finding that the F191R mutation disrupts the interaction of Nef with PAK2 but not its ability to enhance viral replication in ex vivo-infected HLT clearly argues against an important role in cellular activation and in the enhancement of virus production in HIV-1-infected T cells. We also found that disruption of PAK2 binding does not reduce Nef-dependent activation of NFAT in HIV-1-infected Jurkat T cells, providing further evidence that this interaction is not critical for the ability of Nef to render HIV-1-infected cells hyperresponsive to activation. In agreement with the results of our previous study (61), the data presented herein do not support the proposed role of Nef-PAK2 association in the programmed death of virally infected T cells (79).

In sum, we show that residue F191 in HIV-1 Nef is involved in PAK2 binding and enhancement of virion infectivity in P4-CCR5 cells but is not critical for modulation of CD4, MHC-I, and Ii cell surface expression, hyperresponsiveness of HIV-1-infected T cells to activation, efficient infection of TZM-bl cells and PBMCs, stimulation of viral replication in PBMC cultures, or efficient viral spread in ex vivo-infected HLT. Thus, our results do not support a major role of PAK2 interaction in the ability of Nef to enhance viral spread in vivo and to accelerate the progression to AIDS.


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ACKNOWLEDGMENTS
 
We thank Thomas Mertens for support, Daniela Krnavek, Kerstin Regensburger, and Martha Meyer for excellent technical assistance, Ingrid Bennett for critically reading the manuscript, and Gerhard Rettinger, Herbert Riechelmann, Tilman Keck, and Kai-Johannes Lorenz for providing tonsils.

This work was supported by the Medical Research Fund of Tampere University Hospital, the Wilhelm-Sander Foundation, the Deutsche Forschungsgemeinschaft, the Landesstiftung Baden-Württemberg, and NIH grant 1R01AI067057-01A2.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Virology, Universitätsklinikum, 89081 Ulm, Germany. Phone: 49-731-50065109. Fax: 49-731-50065131. E-mail: frank.kirchhoff{at}uniklinik-ulm.de Back

{triangledown} Published ahead of print on 19 September 2007. Back


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Journal of Virology, December 2007, p. 13005-13014, Vol. 81, No. 23
0022-538X/07/$08.00+0     doi:10.1128/JVI.01436-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Laguette, N., Benichou, S., Basmaciogullari, S. (2009). Human Immunodeficiency Virus Type 1 Nef Incorporation into Virions Does Not Increase Infectivity. J. Virol. 83: 1093-1104 [Abstract] [Full Text]  

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