Two Amino Acid Substitutions in the SIV Nef Protein Mediate Associations with Distinct Cellular Kinases

Virology 276, 329–338 (2000) doi:10.1006/viro.2000.0558, available online at http://www.idealibrary.com on

Two Amino Acid Substitutions in the SIV Nef Protein Mediate Associations with Distinct Cellular Kinases Sheila A. Barber,* ,1 Maureen F. Maughan,† ,1 Jason W. Roos,* and Janice E. Clements* ,2 *Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Traylor G-60, Baltimore, Maryland 21205; and †AlphaVax Human Vaccines, Inc., 710 West Main Street, Durham, North Carolina 27701 Received May 8, 2000; returned to author for revision July 24, 2000; accepted July 26, 2000 A functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity. © 2000 Academic Press

notably downregulation of CD4 and MHC I from the surface of infected cells (reviewed in Piguet et al., 1999). Although these functions of Nef have been well characterized in vitro, their relative contributions to viral pathogenesis in vivo have been suggested (Asamitsu et al., 1999; Carl et al., 1999a,b, 2000a,b) but not yet established. Nef also enhances virion infectivity (Flaherty et al., 1997a; Miller et al., 1994) at a postentry event preceding reverse transcription in target cells (Aiken and Trono, 1995; Schwartz et al., 1995), strongly suggesting that optimal infectivity is mediated by virion-incorporated Nef (Pandori et al., 1996; Welker et al., 1996), the majority of which is associated with the mature core (Kotov et al., 1999). We have previously shown that SIV Nef is also incorporated into virions (Flaherty et al., 1997a), but unlike HIV-1 Nef, SIV Nef is not cleaved by the viral protease, an observation functionally consistent with the finding that SIV Nef can enhance the infectivity of nefdefective HIV-1 (Chen et al., 1998), indicating that the ability of Nef to be cleaved by the viral protease does not correlate with increased infectivity. As such, the possibility exists that Nef facilitates virion incorporation of a cellular protein (perhaps activated by the viral protease) that potentiates infectivity. In this regard, Nef has been shown to associate with

INTRODUCTION Although the importance of Nef to viral pathogenesis has been established definitively in vivo in the macaque model of AIDS (Kestler et al., 1991), the critical function(s) of Nef that is interchangeable between HIV and SIV (Alexander et al., 1999; Kirchhoff et al., 1999; Mandell et al., 1999; Sinclair et al., 1997) is still under investigation. Nef is not required for viral replication in vitro, although intriguingly, cis-expression of a dominant negative form of Nef has been shown to inhibit replication of infectious molecular clones as well as clinical isolates (D’Aloja et al., 1998; Olivetta et al., 2000). In infected cells, Nef is found associated with membranes, in the cytoplasm and in the nucleus (Franchini et al., 1986; Guy et al., 1987; Kaminchik et al., 1991, 1994; Kienzle et al., 1992; Ranki et al., 1994; Yu and Felsted, 1992), which suggests that the ability to target different intracellular locations may be important for the complete and apparently versatile function of Nef (Baur et al., 1994). Indeed, several functions of Nef have been described,

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These authors contributed equally to this work. To whom reprint requests should be addressed. Fax: (410) 9559823. E-mail: [email protected]. 2

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FIG. 1. Alignment of amino acid sequences in SIV/17E-Fr and SIV/Fr-2 Nef proteins.

several cellular proteins in a variety of expression systems and cell types. The ability of Nef to associate, in particular, with a p21-activated protein kinase (PAK)-related kinase (Nunn and Marsh, 1996; Sawai et al., 1994), recently identified as PAK-1 (Fackler et al., 2000) or PAK-2 (Renkema et al., 1999), depending on the Nef protein investigated, and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC) (Baur et al., 1997) may correlate with enhanced virion infectivity and proviral DNA synthesis in some cell types (Baur et al., 1997; Luo and Garcia, 1996; Wiskerchen and ChengMayer, 1996). The relevance of these “in vitro Nef-associated kinases” to disease progression in vivo is under investigation, but reported data are controversial (Aldrovandi et al., 1998; Carl et al., 2000b; Khan et al., 1998; Lang et al., 1997; Sawai et al., 1996, 2000). Several potential sites for phosphorylation also exist in Nef that may regulate Nef function (Bandres et al., 1994; Luo et al., 1997a), and a number of reports either confirm that Nef is phosphorylated during infection (Barber et al., 1998) or demonstrate that Nef can be phosphorylated in vitro by cellular kinases such as lck, PKC, and a serine kinase present in NAKC (Bodeus et al., 1995; Coates et al., 1997; Coates and Harries, 1995; Collette et al., 1995; Guy et al., 1990; Luo et al., 1997a). Although the conserved PKC site appears to be important for SIV replication in rhesus macaques (Carl et al., 1999a), the contribution of phosphorylation to Nef function is unknown. In vivo studies of SIV have documented that extensive mutations occur upon inoculation of rhesus macaques that alter the amino acid sequence of Nef (Flaherty et al., 1997b; McArthur, 1987; Sawai et al., 2000; Whatmore et al., 1995), which suggests that accumulated mutations may enable the virus to adapt to rapid changes in intracellular environments, perhaps potentiating productive virus replication in diverse cell types and tissues, including the central nervous system (CNS). To explore this possibility, we first compared in vitro kinase activities associated with Nef proteins derived either from SIV/Fr-2 or from our neurovirulent, molecular clone, SIV/17E-Fr. SIV/Fr-2 is identical to SIV/17E-Fr except that SIV/Fr-2 nef has been replaced with SIVmac239 nef (Flaherty et al., 1997b). The results of our studies indicate that SIV/17E-Fr

causes neurological disease in vivo (Mankowski et al., 1997) despite the fact that the Nef protein of this virus does not activate the PAK-related kinase in vitro (Barber et al., 1998), strongly suggesting that interaction with PAK is not required for virus replication or disease induction in the CNS. Further, we have demonstrated that SIV/ 17E-Fr Nef associates with a novel serine kinase that phosphorylates Nef itself (Barber et al., 1998) and is incorporated into SIV/17E-Fr virions (Flaherty et al., 1997a), potentially playing a pivotal role in virion infectivity. Because there are five amino acid differences between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr (Flaherty et al., 1997b), we examined the contribution of specific amino acids to Nef-associated kinase activity by constructing a panel of clones containing reciprocal reversions of SIV/17E-Fr Nef amino acids to the corresponding SIV/Fr-2 Nef residues. We report that differential kinase associations/activation map to previously uncharacterized amino acids at the N terminus and central core region of SIV Nef. Further, we demonstrate that the essential determinants of Nef-mediated enhancement of viral infectivity are independent of the ability of Nef to associate with any of these cellular kinases. RESULTS Previously uncharacterized amino acids in Nef mediate interactions with different kinases We have previously reported distinct in vitro kinase assay profiles exhibited by the molecular clones SIV/ 17E-Fr and SIV/Fr-2. SIV/17E-Fr, a neurovirulent clone, associates with a novel serine/threonine kinase activity that is incorporated into the virus particle and results in the phosphorylation of Nef itself (Barber et al., 1998; Flaherty et al., 1997a). SIV/Fr-2 is identical to SIV/17E-Fr except that it contains the SIV mac239 nef open gene resulting in five amino acid differences between the two Nef proteins (Fig. 1). The Nef protein of SIV/Fr-2, like SIV mac239 Nef, interacts with a PAK-related kinase (Nunn and Marsh, 1996; Sawai et al., 1994) and a serine kinase in a Nef-associated kinase complex (NAKC) (Barber et al., 1998; Baur et al., 1997). A panel of clones containing reversions of each amino acid in 17E-Fr Nef to the Fr-2

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FIG. 2. (A) In vitro kinase assay of Nef immunoprecipitates prepared from CEMX174 cells transfected with infectious DNA, as described under Materials and Methods, in the presence of 500 ␮Ci/ml [␥- 32P]ATP. A longer exposure of the E(150)K lane is shown at the far right. (B) SDS–PAGE separation of Nef immunoprecipitates prepared from [ 35S]methionine-labeled CEMX174 cells transfected with infectious DNA as described under Materials and Methods. (C) In vitro kinase assays of Nef immunoprecipitates prepared from CEMX174 cells transfected with infectious DNA, as described under Materials and Methods, in the presence of 200 ␮M [␥- 32P]GTP (6000 Ci/mol). Note that the lane locations representing clones K(150)E and E(150)K differ from those in (A) and (B). Results are representative of at least three independent experiments.

sequence was constructed to analyze the contribution of specific amino acids to the differing Nef-associated kinase activities. Nef immunoprecipitates prepared from transfected CEMX174 cells were compared in in vitro kinase assays (Fig. 2A). As previously described, SIV/17E-Fr Nef interacts with a unique serine kinase resulting in the phosphorylation of the 35-kDa Nef protein (Fig. 2A, lane 2), while SIV/Fr-2 Nef exhibits the typical pattern of SIV mac239 Nef-associated kinase activity resulting in phosphorylation of the 62-kDa (autophosphorylated Pak), 97-kDa [likely the recently identified PIX-associated p95 (Brown et al., 1999)] and 35-kDa proteins by the PAKrelated kinase and NAKC (Fig. 2A, lane 3). Substitution of the serine at amino acid position 12 in 17E-Fr Nef with a proline [clone S(12)P] results in the loss of all detectable Nef-associated kinase activity (Fig. 2A, lane 4). The S(12)P Nef protein is equally expressed and immunoprecipitated from 35S-labeled infected cells (Fig. 2B, lane 4); however, specific phosphorylated proteins were never detected in kinase assays of the S(12)P Nef protein. Conversion of the lysine at amino acid 150 in SIV/17E-Fr to a glutamic acid [clone K(150)E] enables detection of the PAK-related kinase activity, as evidenced by the appearance of phosphoprotein (pp) 62 and pp97 without loss of pp35 (Fig. 2A, lane 5). The other amino acid substitutions in SIV/17E-Fr [K(75)E, Q(93)E, and I(119)M] had no effect on the Nef-associated kinase activity (data not shown). Collectively, these results suggest that the

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serine at amino acid 12 in SIV/17E-Fr Nef is required for phosphorylation of pp35 (Nef) or interaction/activation of the serine kinase associated with SIV/17E-Fr Nef, while the glutamic acid at 150 is necessary for interaction/ activation of the PAK-related kinase. To confirm the importance of these specific amino acids to differences in Nef-associated kinase activities, the reverse amino acid substitutions were placed in the background of SIV/Fr-2, and the kinase profiles of these point mutants were compared. The PAK-related kinase activity is conserved in the SIV/Fr-2 P(12)S clone (Fig. 2A, lane 6), while there appears to be increased phosphorylation of pp35 (Fig. 2A, compare lane 6 to lane 3). We consistently observed this increase in phosphorylation, suggesting that the N-terminal serine is a potential phosphorylation site in Nef or that the P(12)S clone has gained a new kinase association that phosphorylates Nef elsewhere. The glutamic acid to lysine substitution in SIV/Fr-2, clone E(150)K, resulted in the loss of the PAKrelated kinase activity as assessed by the disappearance of pp62 and pp97 (Fig. 2A, compare lane 7 to lane 3), and to some extent, NAKC, as evidenced by decreased phosphorylation of pp35 (Fig. 2A, compare lane 7 to lane 3), which appears in longer exposures of the gel (Fig. 2A, side panel). This result confirms the importance of the glutamic acid at amino acid 150 for PAK activation and suggests that PAK activation/interaction may be required for optimal association/activation with the NAKC serine kinase that phosphorylates Nef. The kinase that associates with SIV/17E-Fr Nef utilizes both ATP and GTP as phosphodonors to phosphorylate Nef (pp35), while the SIV/Fr-2 Nef-associated serine kinase (NAKC) utilizes only ATP (Barber et al., 1998). To distinguish the kinases associated with the Nef point mutants in the phosphorylation of pp35, we compared in vitro kinase assays in the presence of [␥- 32P]ATP or [␥- 32P]GTP (Fig. 2C). Consistent with previous results, pp35 is detected in ATP-based assays of both SIV/17E-Fr and SIV/Fr-2 Nef proteins (Fig. 2A, lanes 2 and 3), while pp35 is only detected in GTP-based assays of the SIV/ 17E-Fr Nef protein (Fig. 2C, compare lanes 2 and 3). No phosphoproteins were associated with S(12)P Nef in the GTP-based assay (Fig. 2C, lane 4), again suggesting that this amino acid change results in the loss of all detectable SIV/17E-Fr Nef-associated kinase activity. In both the ATP- and GTP-based assays of K(150)E Nef, pp35 is detected (Fig. 2C, lane 7), indicating that although this Nef gains the PAK-related kinase activity, the SIV/17E-Fr pp35 kinase activity is maintained. The P(12)S Nef-associated kinase also utilizes both ATP and GTP (Fig. 2C, lane 6), confirming that this clone gains the SIV/17E-Fr pp35 kinase activity. Like SIV/Fr-2 Nef, the E(150)K Nefassociated kinase only utilizes ATP to phosphorylate pp35 (Fig. 2C, lane 5), suggesting that this Nef associates with the NAKC but not the SIV/17E-Fr pp35 kinase. Collectively, these results indicate that the serine at

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FIG. 3. SDS–PAGE separation of Nef immunoprecipitates prepared from [ 32P]orthophosphate-labeled CEMX174 cells transfected with infectious DNA as described under Materials and Methods.

amino acid 12 in Nef is required for association/activation of the SIV/17E-Fr-specific kinase or that this serine is the site of Nef phosphorylation by the SIV/17E-Fr-specific kinase. Phosphorylation of Nef is dependent on an N-terminal serine In our first assessment of the potential relevance of in vitro observations of Nef-associated kinase activity to events occurring naturally during viral infection, we previously examined the phosphorylation state of SIV/17E-Fr and SIV/Fr-2 Nef proteins immunoprecipitated from infected CEMX174 cells and macrophages derived from rhesus macaques and reported that SIV/17E-Fr Nef but not SIV/Fr-2 Nef (or PAK) was phosphorylated (Barber et al., 1998). Analogously in this study, we examined the phosphorylation states of Nef proteins immunoprecipitated from [ 32P]orthophosphate-labeled, CEMX174 cells transfected with infectious DNA encoding the SIV/17E-Fr Nef mutants S(12)P and K(150)E or the SIV/Fr-2 Nef mutants P(12)S and E(150)K. Consistent with our previous findings, phosphorylated Nef is detected in lysates of cells infected with SIV/17E-Fr, but not SIV/Fr-2 (Fig. 3, compare lane 2 to lane 3). In fact, we have never detected any of the phosphoproteins (pp62, pp97, and pp35/Nef) routinely observed in in vitro kinase assays of SIV/Fr-2 Nef (Fig. 2A; Barber et al., 1998; Baur et al., 1997; Sawai et al., 1994). Phosphorylation of S(12)P Nef is undetectable even after prolonged autoradiography. Reciprocally, phosphorylation of P(12)S Nef was readily detectable (Fig. 3, lane 6), confirming the requirement of the serine at amino acid position 12 for Nef phosphorylation during infection. Neither the K(150)E mutation in SIV/17E-Fr Nef nor the E(150)K mutation in SIV/Fr-2 Nef affected the phosphorylation state of either parental Nef protein (Fig. 3, lanes 5 and 7). Association with PAK may reduce virion-associated Nef kinase activity An interesting characteristic of SIV/17E-Fr virions is the incorporation of the Nef-associated kinase activity into the virus particle (Flaherty et al., 1997a). To determine whether this kinase activity is affected in the Nef point mutant virions, in vitro kinase assays of Nef immu-

noprecipitates were performed on these virus particles (Fig. 4 top). Consistent with our previous observations (Flaherty et al., 1997a), virus-incorporated Nef kinase activity was readily detectable in SIV/17E-Fr virions. In contrast, both SIV/17E-Fr S(12)P and K(150)E virions contained reduced levels (and in most experiments, barely detectable levels) of virion-incorporated, Nef-associated kinase activity; this is particularly surprising with regard to S(12)P Nef, which exhibited no detectable kinase activity and was not detectably phosphorylated in infected cells. Again consistent with our previous observations, Nef-associated kinase activity was not detected in SIV/ Fr-2 virions (Flaherty et al., 1997a). In contrast, there was detectable Nef-associated kinase activity isolated from SIV/Fr-2 P(12)S virions, albeit a consistently lower level (and at times undetectable) when compared to SIV/ 17E-Fr virions (note that longer exposure times were required for adequate detection). Thus it appears that clones Fr-2/P(12)S and 17E-Fr/K(150)E, the only clones containing both PAK and 17E-Fr-associated kinase activity, exhibit reduced virion-incorporated kinase activity compared to SIV/17E-Fr (compare lanes 2, 5, and 6). Conversion Fr-2/E(150)K had no effect on our inability to detect Nef-associated kinase activity in SIV/Fr-2 virions even after prolonged exposure of autoradiographs. Differences in kinase activity between Nef mutants are not attributable to differential virion incorporation of mutant Nef proteins, as immunoprecipitates of the various Nef mutants from virus produced by transfected, 35S-labeled CEMX174 cells were not notably different (Fig. 4, bottom). Enhanced virus infectivity is not dependent on specific Nef-associated kinases We and others have demonstrated that a full-length Nef protein is required for enhanced viral infectivity (Chowers et al., 1994; Flaherty et al., 1997a; Miller et al., 1995; Pandori et al., 1996; Welker et al., 1996). This increased infectivity may correlate with the ability of Nef to associate with cellular kinases including the PAKrelated kinase, NAKC, and possibly the pp35 kinase

FIG. 4. (Top) In vitro kinase assay of Nef immunoprecipitates prepared from virions isolated from supernatants of CEMX174 cells transfected with infectious DNA as described under Materials and Methods. A longer exposure of the P(12)S lane is shown at the far right. (Bottom) SDS–PAGE separation of Nef immunoprecipitates virions isolated from supernatants of [ 35S]methionine-labeled CEMX174 cells transfected with infectious DNA as described under Materials and Methods.

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FIG. 5. Graphical representation of relative light units (RLU) of luciferase activity produced by LuSIV cells exposed to the various viral clones normalized by TCID50.

associated with SIV/17E-Fr Nef (Baur et al., 1997; Flaherty et al., 1997a; Goldsmith et al., 1995; Luo et al., 1997a). To evaluate the potential contribution of various Nef-associated kinases to viral infectivity, we generated a novel CEMX174/SIVLTR-luciferase indicator cell line (LuSIV) to provide an assay system that closely resembles our infection protocols. In theory, this assay is similar to the MAGI cell assay (Chackerian, 1995) previously utilized by us and others to examine virus infectivity (Flaherty et al., 1997a; Luo et al., 1997a; Goldsmith et al., 1995), and the results of our novel infectivity assay are consistent with these previous reports in that all viruses expressing a full-length Nef protein are more infectious than the SIV/17E-Fr⌬nef virus (Fig. 5). With regard to the mutant viruses described above, viruses expressing SIV mac239 Nef [Fr-2, P(12)S, E(150)K] are, in general, less infectious than viruses expressing SIV/17E-Fr Nef [S(12)P, K(150)E] in this assay. The SIV/Fr-2 P(12)S virus, which appears to associate with PAK, NAKC, and pp35 kinase in in vitro kinase assays, has slightly lower infectivity than SIV/Fr-2 and E(150)K. The loss of the PAKrelated kinase activity does not appear to affect the infectivity of the E(150)K virus. The infectivity of the S(12)P, which has lost all detectable Nef-associated kinase activity, is higher than that of SIV/17E-Fr as is the infectivity of the K(150)E clone, which has retained the pp35 kinase activity and gained PAK association. Similar results were obtained when the input virus was standardized to 50 ng of p27 (data not shown). Collectively, the results of our assay indicate that although a fulllength Nef protein is required for optimal virus infectivity,

there is no correlation with specific in vitro Nef/kinase associations. DISCUSSION Previous reports from this laboratory have described the genetic and biological characteristics of a neurovirulent, molecular clone, SIV/17E-Fr (Edinger et al., 1997; Flaherty et al., 1997b; Mankowski et al., 1994, 1997). The nef gene of SIV/17E-Fr was derived from the neurovirulent strain, SIV/17E-Br, which was obtained originally by serial passage of lymphocyte-tropic SIV mac239 in rhesus macaques (Anderson et al., 1993). Sequence analysis of the nef gene from brain homogenates of a rhesus macaque infected with SIV/17E-Br virus (uncloned) revealed conversion of the proline at position 12 (in SIV mac239) to serine in nine out of nine independent PCR reactions, as well as conversion of the glutamic acid at position 150 to lysine in five of nine independent PCR reactions (M.T.F. and J.E.C., unpublished data). These results support the hypothesis that although functionally tolerable nef mutations evolve rapidly during in vivo passage, which likely enable the virus to adapt to rapid changes in intracellular environments (i.e., cell type and activation state), mutations important for continued growth in certain tissues (i.e., the CNS) will be conserved over time. Genetic evidence supporting the positive selection of nef, in vivo, has been reported (Zanotto et al., 1999). Accordingly, the results of PCR-based sequence analysis described above suggest that (1) there may be selective pressure for conversion to serine at position 12 in brain-derived Nef in vivo and (2) a glutamic acid at position 150 may not

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be absolutely required for neuropathogenesis of SIV in the CNS. In this report, we examined the contribution of specific amino acids that differ between the Nef proteins of SIVmac239 and SIV/17E-Fr. Our results identify two amino acids in Nef, serine at position 12 and glutamic acid at position 150 (both previously uncharacterized with regard to Nef function), that mediate distinct Nef/kinase associations. The presence of glutamic acid at position 150 is conserved without exception in SIV Nef sequences published in Human Retroviruses and AIDS (Korber et al., 1998) and is essential for PAK/Nef interactions, as demonstrated above by the gain of PAK activity associated with K(150)E Nef and the reciprocal loss of PAK activity (but not entirely NAKC activity) associated with E(150)K Nef. As such, the E(150)K clone may well represent the only SIV clone able to associate solely with the serine kinase in NAKC. We have previously reported and confirm in this study that despite the ability of SIV/Fr-2 Nef to associate with PAK and NAKC in cells, SIV/Fr-2 virions fail to exhibit Nef-associated kinase activity. However, mutation of a single amino acid, P(12)S, in SIV/Fr-2 Nef confers detectable virion-incorporated Nef-associated kinase activity concomitant with the ability of P(12)S Nef to associate with the SIV/17E-Fr-associated kinase in cells. Of interest, Nef proteins with the ability to associate in cells with both PAK and the SIV/17E-Fr-specific kinase [P(12)S, K(150)E; Fig. 2], consistently exhibit reduced levels of virion-incorporated Nef-associated kinase activity (Fig. 4). Because virion incorporation of Nef was not influenced by the ability of Nef to associate with cellular kinases, we offer two possible interpretations: (1) Nef proteins complexed with PAK in the cell do not get incorporated into progeny virions and/or (2) the kinase activity present in cellular Nef/PAK complexes is inactivated upon virion incorporation or maturation. The N-terminal amino acids of Nef direct Nef myristoylation and, if expanded to include amino acid 22, suffice for association with the unidentified serine kinase present in NAKC (Baur et al., 1997). The data presented herein extend the functionality of this region in that we demonstrate that the serine at position 12 in SIV/17E-Fr Nef is required both for detection of Nef-associated kinase activity in vitro and for Nef phosphorylation in infected cells. We have previously shown that the kinase associated with SIV/17E-Fr Nef is distinct from the PAKrelated kinase and the unidentified serine kinase in NAKC, due to its unique ability to utilize both ATP and GTP as phosphodonors (Barber et al., 1998). However, we were previously unable to determine definitively if SIV/17E-Fr Nef also associated with the unidentified serine kinase in NAKC. Based on two observations from the data presented above, we are now confident that SIV/17E-Fr Nef does not associate with the unidentified serine kinase in NAKC. First, because the PAK-related

kinase does not phosphorylate Nef (Sawai et al., 1994), we can conclude that the unidentified serine kinase in NAKC, which does phosphorylate Nef, does not require a serine at position 12 because SIV/Fr-2 Nef (expressing a proline at position 12) is phosphorylated in in vitro kinase assays (Barber et al., 1998). Hence, we would expect to see phosphorylated S(12)P Nef in in vitro kinase assays if NAKC was able to associate with SIV/17E-Fr Nef. Second, we always observe a modest increase in the phosphorylation of K(150)E Nef (pp35) in ATP-based in vitro kinase assays of K(150)E Nef with no concomitant loss of GTP-dependent activity (Fig. 2). Since no potential phosphorylation site is provided by the K to E mutation, it seems likely that the mutation enables association with PAK/NAKC. The relevance of Nef/PAK/NAKC associations to Nef phosphorylation during infection in vitro, as potentially related to Nef function(s) during infection in vivo, remains confounding. We have reported previously and present data again here that fail to detect phosphorylated SIV/ Fr-2 Nef or PAK in Nef immunoprecipitates from [ 32P]orthophosphate-labeled SIV/Fr-2-infected cell lysates, whereas cell lysates from parallel SIV/17E-Fr infections contain readily detectable phosphorylated Nef (Fig. 3). Further, mutation of serine 12 abolishes detectable SIV/17E-Fr Nef phosphorylation, an observation consistent with a previous report which demonstrates that 17E-Fr Nef is phosphorylated on serine (Barber et al., 1998). The effect of phosphorylation on Nef function(s) is yet undetermined. Conceivably, phosphorylation of a myristoylated protein may disrupt membrane association, as has been described for both the MARCKS protein and src (reviewed in McLaughlin and Aderem, 1995). Myristoylation of proteins is not sufficient for membrane association, which also requires the presence of exposed basic amino acids to bind acidic phospholipids (McLaughlin and Aderem, 1995). Phosphorylation within the basic cluster can neutralize the association with acidic phospholipids enabling translocation to the cytosol. There are six basic amino acids surrounding (⬃17 amino acids) serine 12 in SIVmac239 and SIV/17EFr Nef proteins, and we are currently evaluating potential differences in the cellular distribution of the mutant Nef proteins. The ability to detect kinase activity associated with S(12)P Nef derived from virions constitutes the most intriguing observation of this study as this Nef protein is not detectably phosphorylated and fails to associate with kinase activity in cells. It is tempting to speculate that the serine at position 12 is the preferred site of phosphorylation by the kinase and that phosphorylation of SIV/17EFr- Nef in virions is so vital for SIV/17E-Fr Nef function (as constricted by the other four mutations) that an alternative phosphorylation site (such as serine at position 13) is utilized under the mutational pressure of S(12)P. Similar sequence flexibilities (i.e., SXXD/E or SXD/E) have

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been described for other kinases such as casein kinase II (Hardie and Hanks, 1995). To evaluate the contribution of Nef-associated kinases to enhancement of virion infectivity, virions representing our panel of Nef point mutants were compared in our novel infectivity assay. The results presented in Fig. 5 show no correlation of particular kinase association with infectivity. In general, SIV/17E-Fr or viruses expressing point mutations in the background of SIV/17E-Fr were more infectious than SIV/Fr-2-based virions. Analyses of the infectivities of the point mutants that did not affect Nef-kinase associations (data not shown) indicated that construction of viruses expressing various combinations of Nef point mutations may be needed to elucidate the basis for increased infectivity of SIV/17E-Fr based virions. As such, we conclude for the SIV/17E-Fr-associated kinase and corroborate the conclusions of independent reports pertaining to other Nef-associated kinases (Luo et al., 1997b; Tokunaga et al., 1999) that although the ability of Nef to associate with cellular kinases may be a conserved feature of Nef, associations between 17E-Fr Nef/unidentified serine kinase and Fr-2 Nef/PAK/NAKC do not translate directly to Nef-mediated enhancement of viral infectivity. MATERIALS AND METHODS Cells CEMX174 cells were obtained from AIDS Research and Reagent Program and maintained in RPMI 1640 (Cellgro, VA) supplemented with 10% FBS, 2 mM sodium pyruvate (Sigma, St. Louis, MO), 10 mM HEPES (Life Technologies, Gaithersburg, MD), 0.5 mg/ml gentamicin (Life Technologies), 2 mM glutamine (Life Technologies); (cRPMI). Construction of Nef point mutants The construction of the infectious molecular clones SIV/17E-Fr and SIV/Fr-2 was described previously (Flaherty et al., 1997b). These clones were used for the construction of the Nef point mutants described here. SIV/17E-Fr contains the nef gene from the neurovirulent virus SIV/17E-Br, while SIV/Fr-2 contains the SIV mac239 nef gene. These two viruses are identical except for the five amino acid coding differences between the two Nef proteins (Fig. 1). To construct the Nef point mutants, the NheI BpuI102I fragments from SIV/17E-Fr or SIV/Fr-2 were amplified by PCR using overlapping primers to mutate the following amino acids in SIV/17E-Fr Nef to the SIV/Fr-2 Nef sequence: K(75)E, Q(93)E, I(119)M, and K(150)E. In SIV/Fr-2 Nef, the E(150)K mutation was made. These sequence modifications do not alter the amino acid sequence of the overlapping transmembrane region of the envelope. The PCR products were digested with SalI and inserted into SalI-digested pLG338/IBI-30.

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The desired mutation and the sequence of the PCR products were confirmed by DNA sequence analysis. The subclones were digested with NheI and BpuI102I, and the fragments were inserted into SIV/17E-Fr or SIV/ Fr-2 to construct full-length molecular clones with the desired amino acid changes. To construct SIV/17E-Fr S(12)P and SIV/Fr-2 P(12)S, the pLG338/IBI-30 NheI–BpuI102I subclones, pLG-17E-Fr and pLG-Fr-2 (Flaherty et al., 1997b) were digested with NcoI and NsiI (amino acids 69–197 in Nef). The purified restriction fragments were swapped between the two clones, resulting in subclones pLG-17E-Fr S(12)P and pLG-Fr-2 P(12)S. The desired mutation and the sequence of the subclones were confirmed by DNA sequence analysis. The subclones were digested with NheI and BpuI102I, and the fragments were inserted into SIV/ 17E-Fr or SIV/Fr-2 to construct full-length molecular clones with the single amino acid change. Transfection and preparation of virus stocks CEMX174 cells were transfected with 10–12 ␮g of infectious DNA by electroporation as described previously (Flaherty et al., 1997a). Cell culture supernatants containing virus were clarified by centrifugation at 1200 g for 10 min, filtered through a 0.45-␮m-pore-size membrane, and quantitated for p27 content, reverse transcriptase activity, and TCID 50 (Flaherty et al., 1997a). Virus stocks were stored at ⫺80°C. Immunoprecipitation and kinase assays Immunoprecipitations and kinase assays were performed as described previously (Barber et al., 1998; Flaherty et al., 1997a). Briefly, transfected CEMX174 cells were collected by low-speed centrifugation, washed once in PBS, and lysed in 300–500 ␮l lysis buffer containing 0.5% (vol/vol) Nonidet-P40, 2 mM EDTA, 137 mM NaCl, 50 mM Tris–HCl (pH 8.0), 10% (vol/vol) glycerol supplemented with protease inhibitors (1 ␮g/ml leupeptin; 1 ␮g/ml aprotinin). Cell culture supernatants containing virus were quantitated by RT assays and clarified twice by centrifugation at 1200 g for 10 min. The clarified supernatants were filtered through a 0.45-␮m-pore-size membrane and concentrated by centrifugation through a 20% (w/vol) sucrose cushion in TNE [25 mM Tris–HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA] at 125,000 g for 1 h at 4°C. The virion pellets were lysed in 300 ␮l lysis buffer (above). Cell and virion lysates were precleared by incubation with normal rabbit serum for 1 h at 4°C followed by incubation with Protein A–Sepharose 4 Fast Flow (Pharmacia) for 1 h at 4°C. The precleared lysates were incubated with 10 ␮l of Nef antisera for 1.5 h at 4°C followed by incubation with Protein A–Sepharose for 1.5 h at 4°C. The immunoprecipitates were washed 3 times with lysis buffer, once with kinase buffer [50 mM

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Tris–HCl (pH 8.0), 10 mM NaCl, 1% Triton X-100, 5 mM MgCl 2, 5 mM MnCl 2, 2 mM sodium orthovanadate], resuspended in kinase reaction buffer [kinase buffer containing 500 ␮Ci/ml [ ␥ -32P]ATP or 200 ␮M [ ␥ -32P]GTP (6000 Ci/mol), ICN Radiochemicals Inc., Irvine, CA], and incubated for 10–12 min at room temperature. The immunoprecipitates were washed with ice-cold lysis buffer and the proteins were analyzed by SDS–PAGE. Metabolic labeling CEMX174 cells were tranfected as described above. Seven to 10 days posttransfection cells were prepared for metabolic labeling. For 35S-labeling, cells were incubated in methionine-free RPMI (Life Technologies) containing 2% FBS and 2 mM glutamine for 1 h before addition of 100 ␮Ci/ml 35S-TransLabel (ICN Radiochemicals Inc.) for 18 h. For 32P-labeling, cells were incubated in phosphate-free RPMI (Life Technologies) containing 10% dialyzed FBS (Life Technologies) and 2 mM glutamine for 1 h before addition of [ 32P]orthophosphate (NEN Life Science Products, Boston, MA) for 18 h. Labeled cells and cell culture supernatants were harvested as described above. Construction of the LuSIV reporter cells The LuSIV reporter cells were constructed using the cell line CEMX174 (permissive for most strains of SIV) and a reporter construct pLuc. The pLuc construct was made by the insertion of the SIV mac239 (⫺225 to ⫹149) long terminal repeat (LTR) upstream of the firefly luciferase reporter gene (obtained in the plasmid pGL3 from Promega, Madison, WI) in the background of the pCEP4 plasmid (Invitrogen, Carlsbad CA). To generate the LuSIV cells, CEMX174 cells (5 ⫻ 10 5) were transfected by electroporation with 12 ␮g of pLuc plasmid DNA using a Bio-Rad gene pulser (Bio-Rad, Richmond, CA). The cells were cultured in cRPMI supplemented with 1 mg/ml hygromycin B to select for stable transfectants. Once a stable cell line was obtained, the hygromycin B concentration was lowered to 300 ␮g/ml for maintenance of the pLuc plasmid. The characterization of these cells is reported elsewhere (Roos et al., 2000). ACKNOWLEDGMENTS The authors thank Debra A. Hauer, David S. Herbst, Brian R. Douglass, Kelly M. Gisselman, and Brandon T. Bullock for their contributions to this work and preparation of the manuscript as well as the rest of the Retrovirus Laboratory for helpful discussions. The work was supported by NIH Grants NS-35751, NS-07392, MH-61189, and HL-061962.

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