Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.


ABSTRACT
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy -termed the "R-clamp" -that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpz P.t.s. ) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpz P.t.t. ) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

AUTHOR SUMMARY
Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein.
Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the Rclamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

INTRODUCTION
Primate lentiviruses comprise human immunodeficiency viruses (HIV-1 and HIV-2), as well as more than 40 simian immunodeficiency viruses (SIVs) infecting non-human primate species from sub-Saharan Africa. HIV-1 groups M, N, O, and P originate from four independent transmissions of SIV from Central chimpanzees (SIVcpz P.t.t. ) and gorillas (SIVgor) to humans, whereas HIV-2 groups A to I are derived from nine zoonotic transmissions of SIV from sooty mangabeys (SIVsmm) (reviewed in [1]). SIVsmm was also transmitted to macaques, giving rise to SIVmac. The global AIDS pandemic is largely caused by HIV-1 group M strains, which are further divided into 10 subtypes (A, B, C, D, F, G, H, J K, and L), as well as several sub-subtypes and circulating recombinant forms [2][3][4].
The viral protein Nef is a multifunctional accessory factor encoded by all primate lentiviruses.
While nef-defective HIV and SIV are replication-competent, infections with such viruses are associated with low viral loads and no or delayed pathogenesis in humans or experimentally infected macaques [5]. Nef itself exhibits no enzymatic activity but instead modulates host cell function by interacting with a plethora of other proteins to hijack cellular signaling and membrane trafficking pathways [6][7][8].
A function of HIV-1 Nef that is well understood at the molecular and mechanistic level is its interaction with Hck, a member of the Src-family tyrosine kinases. Nef tightly binds to the SH3 domain of Hck, thereby pushing it from an intramolecular autoinhibitory state into a catalytically active conformation [9,10]. Hck plays an important role in regulating activation and effector functions of macrophages, the second main target cell population of HIV-1 besides CD4 + T cells [11,12]. Many Nef functions in other cell types including T lymphocytes, (e.g., MHC-I downregulation [13], interfering with Lck localization and immunological synapse formation [13], and reorganization of the actin cytoskeleton [14,15]) are also strictly dependent on its SH3 binding capacity. However, the relevant SH3 proteins in these cells do not bind to Nef as tightly as Hck, and have therefore remained less well characterized [7].
The discovery of the Nef-Hck interaction provided the first example of pathogen takeover of host cell signaling via SH3 domain ligand mimicry [16]. Src Homology 3 (SH3) domains are short (~60 aa) modular protein units specialized for mediating protein interactions via proline-rich core binding sites in the target proteins, and are ubiquitous (~300 in human) in eukaryotic proteins involved in regulation of cell behavior [17,18].
Studies on the Nef-SH3 complex also revealed a new paradigm for the structural basis of SH3 binding: It could be shown that docking of the proline-rich (PxxP) peptide of Nef by the SH3 core binding interface is assisted by further molecular contacts of the so-called SH3 RT-loop region to provide additional specificity and affinity to this interaction [9]. The amino acid residues that form a binding pocket for the RT-loop of the Hck-SH3 domain are highly conserved in HIV-1 and HIV-1-like Nef proteins, and provide them with a capacity for strong binding to Hck [9,19]. Nef proteins from the HIV-2/SIVsmm groups lack these residues and hence Hck binding capacity, but as evidenced by the SIVmac293-Y113W/E117T/E118Q triple mutant [20], can acquire this function upon introduction of just a few key residues from HIV-1 Nef.
We have recently shown that Hck activation by Nef leads to Raf/MAPK pathway activation and triggers the secretion of proinflammatory cytokines [19,21]. In agreement with their enhanced affinity for Hck [9,20], we found that only HIV-1/SIVcpz type Nef proteins but not HIV-2/SIVsmm type Nef proteins show this function. The current study was incited by our observation that an HIV-1 group N Nef (clone YBF30) was unable to bind and activate Hck and the Raf/MAPK pathway for reasons that could not be readily explained by its amino acid sequence. Our investigation into this issue led to the discovery of a structural arrangement that we have termed the arginine R-clamp and characterized here. We show that different but functionally equivalent R-clamps have emerged in the HIV-1/SIVcpz lineage to coordinate Hck SH3 domain binding by Nef. This finding further highlights the importance of Nef-mediated SH3 binding and the enormous plasticity of primate lentiviral accessory proteins.

RESULTS
In our previous studies we examined the capacity of Nef to activate Hck by monitoring tyrosine phosphorylation of paxillin, a prominent substrate for Src family tyrosine kinases, and by measuring the induction of AP-1-regulated reporter gene expression following Hckactivated MAPK signaling [19,21]. When additional Nef proteins from HIV-1 groups N, O, and P were examined (Figure 1), we unexpectedly noticed that the Nef protein of HIV-1 N YBF30 (AJ006022) was unable to induce paxillin phosphorylation or AP-1 activity despite sharing the 72 PxxPxR 77 motif (numbering based on the HIV-1 Nef consensus) known to be important for Hck binding by HIV-1/SIVcpz type Nefs ( Figure 2). Importantly, YBF30 Nef was expressed well in these cells ( Figure 1A), and as also shown previously [22] was fully functional when tested for its capacity to downregulate cell surface expression of CD4 ( Figure   1C and Supplementary Figure 1).
To examine if this is a characteristic of several HIV-1 group N viruses we tested three additional HIV-1 N Nef proteins from the strains 02CM-DJO0131 (AY532635), YBF106 (AJ271370), and S4858 (KY498771). However, we found that all of them were fully competent for Hck activation, and induced paxillin phosphorylation and AP-1 activity as efficiently as Nef from the widely used HIV-1 group M strain SF2 ( Figure 1).

B.
A.
HIV-1 Nef consensus sequence: Since the core SH3 docking motif ( 72 PxxPxR 77 ) as well as the key RT-loop accommodating residues, including Phe90, Trp113, Thr117, and Gln118, were conserved in YBF30 Nef (see Figure 2) we had a more detailed look at the original X-ray structure of the HIV-1 Nef-SH3 complex (1EFN; [9]), which directed our attention to Nef residue 120. This position is occupied by an aromatic residue (tyrosine or phenylalanine) in virtually all HIV-1 M Nef proteins, while an isoleucine residue is found in this position in YBF30 Nef ( Figure 2). (C) In the Y120I structure, one side of the R77 guanidinium plane is lacking non-bonded interactions.
(D) In the A83M/Y120I, structure the long methionine side chain re-establishes non-bonded contacts for R77. The presented mutated complex structures were created by replacing Y120 or Y120 and A83 in UCSF Chimera [23] and minimizing the structures with AMBER [24].
In the 1EFN structure, Y120 can be seen to coordinate the Nef-SH3 interaction by buttressing the side-chain of the R77 in the Nef 72 PxxPxR 77 motif together with the SH3 residue W37 (SH3 numbering according to [18]) in order to stabilize the close positioning of R77 with the acidic SH3 residue D17 to form a critical salt bridge with it ( Figure 3A and B). We termed this steric guiding of the Nef residue R77 into close proximity of D17 in Hck-SH3 the "arginine (R)clamp", and hypothesized that the failure of YBF30 Nef to activate Hck is due to the failure of its Ile120 residue to from a functional R-clamp ( Figure 3C).
In support of this hypothesis, we found that YBF30 Nef became fully competent for Hck activation when its Ile120 residue was replaced by a tyrosine (YBF30 I120Y) ( Figure 1). We then further investigated this idea by changing the Y120 residue in the Nef protein from the HIV-1 M laboratory strain SF2 into an YBF30-like isoleucine (Nef mutant SF2 Y120I) or into a phenylalanine, the other commonly found residue at this position (Nef mutant SF2 Y120F).
While the conservative Y120F amino acid change did not affect Hck activation, the Y120I mutation recapitulated the failure of YBF30 Nef to stimulate paxillin phosphorylation and AP-1 activity, without compromising protein stability or CD4 downregulation ( Figure 1).
Thus, we conclude that the lack of a functional R-clamp due to the I120 residue likely explains the inability of YBF30 Nef to activate Hck.
Analysis of additional HIV-1 group N Nefs revealed that in 5 out of the total of 12 sequences that are available in databases the position 120 was occupied by Ile (Table 1). This is in stark contrast to the Nef proteins from the three other HIV-1 groups M, O, and P, in which residue 120 is invariably Tyr or Phe. We had access to another Ile120-containing HIV-1 N Nef namely 2693BA (GQ925928), and went on to examine its ability to activate Hck. Having already established a strict correlation between paxillin phosphorylation and AP-1 activity as markers of Hck activation ( Fig. 1 and Zhao et al 2021), we chose to focus on using AP-1-driven reporter gene expression as the read-out in our further studies. Unexpectedly, we found that despite carrying the I120 residue 2693BA Nef had an undiminished capacity to activate Hck ( Figure   4). In order to understand this finding, we returned to the Nef-SH3 structure 1EFN, and noted the involvement of Nef residue 83 in contacting the critical R-clamp residue 120 ( Figure 3).
Residue 83 is an amino acid with a tiny (Ala or Gly) or a hydrophilic side-chain (Gln, Ser, Asp, or Glu) in virtually all Nef proteins that have an aromatic (Phe or Tyr) residue at position 120. However, in 2693BA Nef position 83 is occupied by methionine, a residue with a large hydrophobic/aliphatic side-chain. We therefore hypothesized that M83 might compensate for the presence of isoleucine instead of a planar aromatic residue at position 120 ( Figure 3D and Supplementary Figure 4), and that the M83/I120 residue pair of 2693BA Nef might be functionally equivalent to the A83/F120 pair found in the Hck-activating HIV-1 N Nef proteins 02CM-DJO0131 and YBF106. To test this idea, we swapped residues 83 between the I120-containing 2693BA and YBF30 Nef proteins. In line with our hypothesis, this reversed the differences in the Hck-activating capacity of these HIV-1 N Nefs, such that Nef-2693BA lost its ability to stimulate AP-1 activity, whereas YBF30 Nef became fully competent for this function (Figure 4). Thus, we conclude that Nef residues 83 and 120 appear to function together to form the R-clamp that coordinates Hck-SH3 binding by Nef, and that different combinations of residue pair 83/120 have evolved for building a functional R-clamp in HIV-1 group N Nefs.
Whereas among the Nef proteins of HIV-1, Ile can be found at position 120 only in group N viruses, we noted that several SIVcpz Nefs also contain Ile120 ( , whereas both of the two non-activating SIVcpz Nef proteins contained a mixed pattern of amino acid classes at these positions (Figures 2 and 5). To further prove this concept, we showed that the failure of Cam5 and EK505 Nef to activate Hck could indeed be corrected by introducing a tyrosine residue at position 120 in these CD4-AF647 fluorescence hydrophilic residue at position 83 of Nok5 Nef while maintaining its Ile120 residue (Nok5 I83Q) led to a complete loss of its Hck-activating potential, as predicted by our model on the compatible and non-compatible R-clamp residue combinations.  When the residue 83/120 composition was analyzed in a comprehensive survey of HIV-1/SIVcpz/SIVgor Nef sequences, it was interesting to observe that the different evolutionary lineages of primate lentivirus are associated with distinct R-clamp design strategies. In Table 1 Based on the experiments presented above it was inferred that Nef proteins in categories I, II, and IV can bind Hck to stimulate its tyrosine kinase activity, whereas category III Nef proteins with a "mixed" R-clamp residues pattern fail to do so. To further substantiate the generality of this conclusion, we introduced several additional individual and combinatorial 83/120 residue changes into the model Nef protein of HIV-1 M SF2 according to the classification presented in Table 1. We found that a Gln, Glu, or Ser residue could indeed all be introduced at position 83 of SF2-Nef without losing its Hck-activating function, as long as an aromatic residue was maintained at position 120 ( Figure 6; SF2 A83Q, A83E, and A83S).
Thus, all these different category II configurations could support a functional R-clamp when artificially introduced into SF2-Nef.

DISCUSSION
Apart from the regulation of trafficking of CD4 and other membrane proteins, the majority of all cellular functions described for Nef depend on its SH3 domain binding capacity, and are lost if the highly conserved consensus SH3 ligand motif PxxPxR of Nef is mutated [7].
Here we show that Nef proteins of HIV-1 and closely related SIVs have evolved a sophisticated molecular mechanism that we have termed the R-clamp in order to coordinate their SH3 binding. This term was coined due to buttressing of the arginine residue of the Nef PxxPxR motif (R77) by the coordinated action of the Nef residues 83 and 120 together with a tryptophan residue (W37) that is conserved in almost all SH3 domains. This places R77 of Nef into close proximity with another highly conserved SH3 residue (D17) to form a salt bridge that stabilizes the Nef/SH3 complex. Thus, docking of proline-rich ligand peptides presented by native proteins, such as Nef, can involve an additional level of structural complexity for tuning of SH3 binding affinity and specificity that is not evident from studies with isolated SH3 binding peptides.
Our studies have focused on the SH3-mediated interaction between Nef and the tyrosine kinase Hck leading to enzymatic activation of Hck, which we have monitored based on induction of downstream signaling events, including paxillin phosphorylation as well as triggering of the Raf/MAPK cascade and subsequent AP-1-regulated gene expression.
Nef/Hck interaction is thought to play an important role in HIV infection of cells of the myeloid lineages, such as macrophages, and is an especially tractable study system because of the high affinity of Hck SH3 domain for Nef [19]. This is due to the additional affinity brough into this interaction by tertiary contacts outside of the canonical SH3 ligand binding surface and involving the tip of the RT-loop in Hck SH3 [9,25]. Because of the lack of such RT-loop contacts, SH3 domains of other host cell proteins bind to Nef with a lower affinity resulting in more transient interactions that have remained less well characterized, but appear to include at least Lck [26], Vav [27], PACS-1 [28], Btk, and Itk [29]. Nevertheless, similar to Hck-binding, all SH3 interactions by Nef are expected to depend on formation of a salt bridge between R77 of Nef and acidic (D or E) SH3 residue 17. Thus, all these interactions, including the SH3-mediated complexes that are relevant for Nef in T lymphocytes depend on a functional R-clamp.
Interestingly, we found that in different lentiviral Nef proteins the R-clamp has been assembled based on alternative designs, which depend on the combination of amino acids at positions 83 and 120. Moreover, the preferred 83/120 residue combinations are dissimilar in different lineages of HIV-1, SIVcpz, and SIVgor (Table 1) comprises category I and III Nefs, it is tempting to speculate that the zoonotic transmission that gave rise to HIV-1 group N involved a virus carrying a Nef with a category III R-clamp.
In line with this hypothesis, the inferred common ancestor of HIV-1 group N viruses belongs to category III as its Nef protein harbors a combination of Q83 and I120 [30](GenBank accession: KP059120.1). Furthermore, the SIVcpz(P.t.t.) isolate EK505 (DQ373065), a close relative of HIV-1 group N viruses [31] also expresses a category III Nef. Thus, HIV-1 group N viruses may all be the result of a category III virus, and still be adapting and evolving into categories I and IV with functional R-clamps. Notably, group N most likely represents the evolutionarily youngest of all HIV-1 groups [32,33], and the three most recently isolated group N Nefs (JN572926, MF767262, KY498771), including that of N1_FR_2011, the only group N virus isolated outside Cameroon [34], belong to categories I or IV. A single amino acid change of residue 83 (like the EK505-E83M mutant created in this study; see Fig. 5) would have been sufficient to move from category III to IV, whereas the path from category III to I would require changes in both residues 83 and 120, and thus follow an evolutionary trajectory via category II. None of the currently available HIV-1 N Nef sequences fall into the R-clamp category II, but given the scarcity (n=12) of these sequences this does not mean that such HIV-1 N Nefs could still not exist. KP059119.1; [30] belongs to category I. This is also true for the oldest known HIV-1 M Nef (M15896) isolated in 1976 [35]. Finally, the exclusive use of category I R-clamp by Nef proteins of SIVgor, HIV-1 O, and HIV-1 P would suggest that an R-clamp category I virus might have originally been transmitted from chimpanzee to gorilla, and subsequently in two independent cross-species jumps entered humans, giving rise to HIV-1 O and HIV-1 P.
It is of interest to note that a subtype D virus of the HIV-1 M group has been previously shown to encode a Nef protein unable to activate Hck, and that this deficiency was mapped to the unusual presence of an isoleucine residue at the Nef position 120 [36]. This study suggested that Tyr120 would contribute to the hydrophobic pocket of Nef that accommodates the Hck SH3 domain RT-loop, whereas I120 could not serve this function. However, these molecular contacts inferred based on our (NL4-3) Nef/SH3 X-ray structure (1EFN; [9]}) would be expected to only minor rather than critical for the Nef-Hck interaction, and a recent SH3 complex structure involving HIV-1 SF2 Nef [36] does not support such a role for Y120 at all. Moreover, our current data clearly show that category IV Nef proteins containing I120 do bind and activate Hck when paired with an appropriately matched R-clamp residue at the position 83. Thus, while the findings of Choi and Smithgall on Nef from HIV-1 ELI [36] are in agreement with our data, they need to be interpreted in light of the R-clamp concept described here.
While the evolutionary scenarios discussed above may help to explain the occurrence of inactive category III Nefs in HIV-1 group N, it still remains unclear why about one third of all SIVcpz(Pt.t.) Nefs harbor a category III R-clamp. Furthermore, the reasons for the unique predominance of category IV Nefs in SIVcpz(P.t.s.) remain to be determined. Apart from the initial founder viruses that gave rise to the respective lentiviral lineages and species, the optimal R-clamp composition and the rate of R-clamp sequence evolution are probably determined by a complex balance between host-specific factors and sequence variation elsewhere in these viruses. To gain further insights, it would be interesting to examine a larger number of HIV-1 N Nef sequences over time and from different tissues from the same individual to understand whether the R-clamp pattern is relatively fixed or subject to rapid evolution and quasispecies variation. In the latter case, because of the highly multifunctional nature of the Nef protein, a non-functional R-clamp might provide a selective advantage in certain circumstances or anatomic locations. On the other hand, considering that an intact SH3 binding motif PxØPxR (where Ø is a hydrophobic residue, almost invariable a valine) is universally conserved, including Nef proteins with a category III R-clamp, it is also possible that while unable to interact with canonical SH3 domains, these Nef proteins might instead have an unusual specificity for certain atypical SH3 proteins, which could provide them with alternative cellular functions.
In any case, R-clamp sequence variation provides a fascinating example of evolutionary plasticity of a protein interaction interface and our findings suggest that the selection pressures that have shaped Nef during primate lentiviral evolution are different depending on the HIV/SIV lineage and their host species. The dynamic capacity of Nef for altering the molecular strategy of recognizing its key host cell interaction partners, such as SH3 domains, is also important to keep in mind in attempts to develop novel HIV eradication therapies that might target the immune evasion function of Nef.

Reagents and cell lines
Mouse anti-Myc (sc-40) and rabbit anti-paxillin (sc-5574) were from Santa Cruz Biotechnology. Mouse anti-pY31 paxillin was from BD Biosciences. Alexa Fluor 647 conjugated anti-human CD4 antibody was from SouthernBiotech. IRDye680CW goat antimouse IgG, and IRDye800CW goat anti-rabbit IgG were from Li-Cor Biotechnology. HEK293 cells and Jurkat cells were obtained from ATCC. The derivation and characterization of the HEK293-based Hck-expressing HZ-1 cells have been described elsewhere [19]. HEK293 and HZ1 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS), 0.05 mg/ml penicillin, and 0.05mg/ml streptomycin. Lentiviral transduction was employed to generate Jurkat cells stably expressing human CD4. Briefly, HEK293 cells were co-transfected with 2.5 !g pDelta8.9, 1.5 !g VSV-G and 3 !g pWPI-puro plasmid containing human CD4 cDNA in Opti-MEM medium with 12 !g PEI. After 5 hours, medium was refreshed with cell culture media.
Supernatant was collected 48 hours post-transfection, filtered and used to infect Jurkat cells.
Infected cells were selected with 6 !g/ml puromycin for 2 days. Single cell clones of CD4 transduced Jurkat cells were isolated by cellenONE X1 system. Jurkat cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS, 0.05 mg/ml penicillin, and 0.05 mg/ml streptomycin.

Immunoblots
Cells were collected and lysed on ice for 10 minutes in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% NP-40) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 16,000 x g at 4°C for 5 min. Proteins from cell extracts were analyzed by standard SDS gel electrophoresis and Western blotting using IRDye-labeled detection reagents detailed above.

AP-1 luciferase reporter assay.
HZ1 cells were transfected using TransIT-2020 reagent (Mirus) with 50 ng of Nef expression vector together with 50 ng of AP-1 pfLUC reporter plasmid [37] driving the AP-1 inducible expression of firefly luciferase, plus 50 ng of the plasmid pRL-TK (Promega) expressing low and constitutive levels of Renilla luciferase. Cells were collected and lysed with lysis buffer (Promega) on ice. A dual-luciferase reporter assay system from Promega was utilized to determine luciferase activities following the manufacturer's protocol using Berthold Sirius single-tube luminometer detection.

Transduction of Jurkat cells and flow cytometry analysis of CD4
Nef-expressing HIV-1-like pseudoviruses were produced as described previously [19]. Jurkat cells stably expressing CD4 were infected with such lentiviral vectors containing various nef