Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein*

Background: The molecular details of the TRN-SR2/HIV-1 IN interaction are not known. Results: Crucial amino acids in IN for the interaction with TRN-SR2 are located in the CTD, Arg-262/Arg-263/Lys-264, and Lys-266/Arg-269. Conclusion: TRN-SR2 primarily interacts with the CTD domain of IN. Significance: Understanding of the IN/TRN-SR2 interaction is necessary to guide drug discovery efforts targeting the nuclear entry step of replication. Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP1, amino acids 170EHLKTAVQMAVFIHNFKRKGGI191) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids 214QKQITKIQNFRVYYR228 and 262RRKVKIIRDYGK273) that come together in the CTD tertiary structure to form an exposed antiparallel β-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication.

The human immunodeficiency virus type 1 (HIV-1) belongs to the family of Lentivirinae, characterized by the ability to infect both dividing and nondividing cells (1,2). After virus entry and reverse transcription, the preintegration complex (PIC) 5 translocates to the nucleus, and the viral DNA is integrated into the host chromatin. HIV crosses the nuclear membrane through active nuclear import of the PIC (3). As efficient integration into human chromatin depends on nuclear import, and only few PICs seem to enter the nucleus, this process constitutes a true bottleneck for viral replication (4). Until recently, the mechanism of HIV nuclear import, albeit intensely studied, remained obscure (for recent reviews see Refs. 5,6). Several viral elements, including matrix, viral protein R, capsid, IN, and the central polypurine tract have been suggested to play a role in the nuclear import of HIV (5,(7)(8)(9)(10). Next to different viral components, multiple nuclear import factors have been implicated in the nuclear translocation of the PIC, such as importin-␣/␤ (11)(12)(13), importin-7 (14 -16), and importin-␣3 (17). Recently, transportin-SR2 (TRN-SR2, TNPO3) has been proposed as a cellular cofactor of HIV-1 replication (18 -20) mediating nuclear import of the PIC (19,20). Independent of two genome-wide siRNA screens (18,20), TRN-SR2 was identified as an interaction partner of HIV-1 integrase in a yeast twohybrid screen (19). A reverse screen confirmed the interaction and demonstrated that none of the other HIV proteins directly interact with TRN-SR2. Using RNAi technology, the crucial role of TRN-SR2 in HIV replication was demonstrated (18 -20). A specific reduction in two long terminal repeat (LTR) circles and integration was measured by quantitative PCR (Q-PCR) (19,21,22). Direct evidence for TRN-SR2-mediated nuclear import of HIV was obtained by using fluorescently labeled PICs (19). The importance of TRN-SR2 for HIV replication has been confirmed by multiple reports, but its exact mechanism of action is still under debate (7, 18 -28). HIV-1 integrase (IN) mediates the integration of the viral DNA into the host chromatin (29). It consists of three domains connected by flexible linkers as follows: 1) the N-terminal HH-CC zinc-binding domain (NTD); 2) the catalytic core domain (CCD), and 3) the C-terminal domain (CTD) (30). Integrase has been implied in nuclear import of HIV in multiple studies, and various nuclear localization sequences have been reported. First, a bipartite nuclear localization signal in the CTD was described that consisted of amino acids 211 KELQKQITK 219 and 261 PRRKAK 266 (11). Later, a nonclassical NLS 161 IIGQVRDQAEHLK 173 was described in the CCD (31). Although TRN-SR2 was identified in a yeast two-hybrid screen as an interaction partner of HIV-1 IN (19), the question still remains whether the interaction between IN and TRN-SR2 mediates HIV nuclear import. As a first step in answering this question, we analyzed the interaction of TRN-SR2 and HIV-IN in molecular detail to identify the minimal interacting regions and the protein-protein interaction hot spots in HIV IN.

EXPERIMENTAL PROCEDURES
Recombinant Protein Purification-Recombinant proteins were expressed in Escherichia coli BL21-CodonPlus (DE3). Recombinant His 6 -tagged HIV-1 integrase was purified as described previously (32). We thank Dr. Woan-Yuh Tarn (Institute. of Biomedical Sciences, Taiwan) for the pGEX-TRN-SR2 expression plasmid. Recombinant GST-tagged and Histagged TRN-SR2 were purified as described previously (19). For the expression of the GST peptides, bacteria were grown to an OD of 0.6, and protein expression was induced with 0.5 mM isopropyl ␤-D-thiogalactopyranoside. After incubation at 37°C for 2 h, the bacteria were harvested, washed, and stored at Ϫ20°C. For purification of the GST peptides, the cells were resuspended in lysis buffer (PBS (pH 7.4), 0.5 M NaCl, 1 mM DTT, 1 mg/ml lysozyme, 0.1 mM PMSF, 1 l of DNase/10 ml). After complete lysis by sonication, the supernatant was cleared by centrifugation, and recombinant proteins were bound to glutathione-Sepharose resin (GE Healthcare). After washing of the resin with 20 volumes of washing buffer (PBS (pH 7.4), 0.5 M NaCl, 1 mM DTT), the GST-tagged protein was eluted with 10 ml of elution buffer (PBS (pH 7.4), 0.5 M NaCl, 1 mM DTT, 20 mM reduced glutathione). The fractions were analyzed by SDS-PAGE for protein content, pooled, and dialyzed (overnight, 4°C) against storage buffer (PBS (pH 7.4), 1 mM DTT, 10% (v/v) glycerol).
AlphaScreen Binding Assay-The AlphaScreen binding assay was optimized for use in 384-well OptiPlate microplates (PerkinElmer Life Sciences) with a final volume of 25 l. Proteins were all diluted to 5ϫ working solutions in the assay buffer (25 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl 2 , 2 mM DTT, 0.1% (v/v) Tween 20, and 0.1% (w/v) bovine serum albumin (BSA)). First, 10 l of the TRN-SR2 was pipetted into the wells, followed by 5 l of His 6 -IN or a GST-peptide dilution series. The plate was sealed and left to incubate for 1 h at 4°C. Next, 10 l of a mixture of Ni 2ϩ chelate acceptor and glutathione donor AlphaScreen beads (PerkinElmer Life Sciences) was added. This establishes final concentrations of 20 g/ml for each of the beads. Plates were then incubated for 1 h at 30°C and analyzed using an EnVision Multilabel Reader (PerkinElmer Life Sciences) according to the manufacturer's instructions. Each titration was performed in duplicate, and assays were repeated at least twice in independent experiments. The equilibrium dissociation constants (apparent K d ) were calculated with Prism 5 (GraphPad), by plotting the titration curves using a one-sitespecific binding with Hill slope fit as shown in Equation 1, The background signal was subtracted from each value. Points for which quenching of the signal occurs through excess of either binding partner ("hooking" as referred to by the supplier), were excluded from the final plot.
Integrase Activity Assays-The 3Ј-processing activity was measured in a standard assay using radioactively labeled oligodeoxynucleotide substrates resembling the viral LTR end, although a quantitative ELISA was used to measure the overall enzymatic activity (33).
Viruses and Viral Vectors-The viral molecular clones pNL4-3 and pNL4-3.Luc.R-EϪ were obtained through the AIDS Research and Reference Reagent Program. The pMD.G plasmid encodes the vesicular stomatitis virus glycoprotein (34,35). Viruses were produced by single polyethyleneimine-mediated transfection of 293T cells using 20 g of the viral molecular clone pNL4-3 per 10-cm dish. Vesicular stomatitis virus glycoprotein-pseudotyped viral vectors were produced by double polyethyleneimine-mediated transfection of 293T cells using 5 g of the envelope expression plasmid pMD.G and 20 g of the molecular clone pNL4-3.Luc.R-EϪ per 10-cm dish. Two and 3 days post-transfection, the supernatant was harvested, filtered with 0.22-m pore-size syringe filters (Sartorius), concentrated by centrifugal filtration using VivaSpin concentrators (Millipore), and treated with DNase I (Roche Applied Science).

Determination of the Interaction Domain between HIV-1
Integrase and TRN-SR2-By yeast two-hybrid, we previously identified TRN-SR2 as a bona fide binding partner of HIV-1 IN (19). A reverse screen confirmed the interaction between TRN-SR2 and IN and demonstrated that no other viral protein interacts with TRN-SR2 under these conditions. By now, the inter-
To define the minimal TRN-SR2 interaction domain in HIV-1 integrase, we now investigated its interaction with the NTD, the CCD, and the CTD. The different IN domains fused to GFP were expressed in 293T cells, and TRN-SR2 was expressed with a 3ϫFLAG tag (Fig. 1A). By co-IP, TRN-SR2 was shown to interact with both the catalytic core and the C-terminal domain of IN but not with the NTD (Fig. 1A). Next, deletion mutants of either the CCD or the CTD were generated and tested for interaction with TRN-SR2 in co-IP (data not shown). This approach failed to generate further insights because mutants bound nonspecifically to the agarose beads, most likely due to their reduced structural stability. We therefore opted for an in vitro approach.
We purified recombinant full-length IN and its domains carrying an N-terminal His 6 tag as well as recombinant TRN-SR2 with an N-terminal GST tag to determine the interaction by AlphaScreen (Fig. 1B). The apparent K d value as well as the AlphaScreen counts are important to compare the affinity of two proteins tested in AlphaScreen. Both the CCD and the CTD of HIV-1 IN displayed affinity for TRN-SR2. The CCD of IN interacts with TRN-SR2 with a 2-fold lower affinity (K d ϭ 40.2 Ϯ 15.1 nM) and 10-fold lower counts than full-length IN (K d ϭ 16.8 Ϯ 3.8 nM), whereas the CTD of IN interacts with TRN-SR2 in a cooperative manner with a 6-fold lower affinity (K d ϭ 94.2 Ϯ 2.6 nM) but 4-fold higher counts than full-length IN (Fig. 1B). The data confirm direct protein-protein interactions between TRN-SR2 and both CCD and CTD of HIV-1 IN as shown by co-IP. Hence, we focused our subsequent analysis of the interaction interface on these two domains.
Defining the Minimal Interaction Domain in the CCD of HIV-1 IN-Nuclear localization signals are usually composed of short amino acid stretches and are commonly bipartite (36). Classical NLSs for instance are known to be composed of one or two basic-rich stretches (37). Therefore, single point mutations usually do not abolish the interaction of a cargo with its specific nuclear import factor (38, and multiple mutations are required. Likewise, abolishing the IN/TRN-SR2 interaction by single point mutations proved to be difficult in our hands, and deletion mutants of IN domains displayed poor solubility. 6 Therefore, we opted for a different strategy, selecting short oligopeptide stretches that are lining the surface of the CCD of HIV-1 IN and screened those for interaction with TRN-SR2. For ease of expression and purification, we produced the small CCD fragments with a GST tag and analyzed their binding to His 6 -TRN-SR2. Reversal of the affinity tags did not affect the interaction of IN with TRN-SR2 (data not shown; GST-IN/His 6 -TRN-SR2, K d ϭ 18.6 Ϯ 1.9 nM; His 6 -IN/GST-TRN-SR2, K d ϭ 16.8 Ϯ 3.8 nM). We purified four distinct GST-tagged oligopeptide fragments representing the four ␣-helices lining the CCD surface (Table 1) and analyzed them for their affinity to His 6 -TRN-SR2 ( Fig. 2A).
One of the four peptides (CCD(170 -191)) displayed a moderate affinity for His 6 -TRN-SR2 ( Fig. 2A). The AlphaScreen signal for CCD(170 -191) allowed us to determine a K d of 401.4 Ϯ 7.7 nM. We will refer to the CCD(170 -191) peptide as integrase-derived interaction peptide 1 (INIP 1 ). To map the amino acids involved in the interaction, we performed mutagenesis of selected residues in INIP 1 . The selection of mutants was guided by the general knowledge of NLS sequences and the surface exposure of amino acid residues in integrase (Fig. 2B). Therefore, we generated two mutants of INIP 1  ) and assayed their affinity for TRN-SR2 (Fig. 2C). Both mutants of INIP 1 displayed a reduced affinity for TRN-SR2. To confirm that these amino acids were the main contributors for the IN-TRN-SR2 interaction, we generated the corresponding mutants in the CCD domain (CCD E170Q/H171A/K173A and CCD F185A/K186A/R187A/K188A ). In agreement with the results obtained with the peptides, CCD E170Q/H171A/K173A showed a reduced affinity for TRN-SR2, although CCD F185A/K186A/R187A/K188A did not interact any longer with TRN-SR2 in AlphaScreen (Fig. 2D).
Defining the Minimal Interaction Domain in the C-terminal Domain of HIV-1 IN-Following the same approach, we divided the C-terminal domain of HIV-1 IN into short oligopeptide stretches of 15 amino acids each (Table 1) and screened those for interaction with TRN-SR2. The small CTD fragments were purified with a GST tag. The five distinct GST-tagged oligopeptide fragments together covered amino acids 214 -288 of the CTD and were analyzed for their affinity to His 6 -TRN-SR2 in the AlphaScreen assay (Fig. 3A).
Mapping of the Hot Spots of Interaction in INIP 2 -To pinpoint the actual protein-protein contacts, we performed mutagenesis of selected residues in INIP 2 . Selection of mutants was again based on the general knowledge of NLS sequences and amino acid surface exposure in IN (Fig. 4A). Because a typical NLS contains a set of basic residues, we first generated mutant INIP 2 versions in which two or three basic residues were substituted with alanine. Some NLSs, like the M9 NLS (37), are rich in glycine and aromatic residues. Hence, we also mutated several aromatic residues into alanine. We then tested these mutant peptides for interaction with TRN-SR2 in the AlphaScreen assay (Fig. 4B) (Fig. 4B). The binding of INIP 2 F223A/Y226A was too weak to allow confident K d determination.
As none of the initial substitutions led to a complete loss of binding, we generated two CTD mutants each containing two sets of substitutions generated in INIP 2 (CTD F223A/R224A/Y226A/R228A and CTD R262A/R263A/K264A/K266A/R269A ). CTD F223A/R224A/Y226A/R228A showed a reduced affinity for TRN-SR2, although CTD R262A/R263A/K264A/K266A/R269A completely lost binding to TRN-SR2 in AlphaScreen (Fig. 4C). To narrow down the number of substitutions needed to abrogate the binding of the CTD to TRN-SR2, we generated two more CTD mutants (CTD R262A/R263A/K264A and CTD K266A/R269A ). CTD K266A/R269A showed a reduced affinity for TRN-SR2 although CTD R262A/R263A/K264A completely lost binding to TRN-SR2 (Fig. 4C).  The fragment numbers refer to the positions of the amino acids in integrase and define the first and the last amino acid of each CTD fragment. The peptides were designed to match protein stretches (15-22 amino acids) of IN with at least some surface-exposed amino acids.

HIV-1 IN C-terminal Residues Interacting with TRN-SR2
with WT IN (Fig. 5A). The F185A/K186A/R187A/K188A substitutions only moderately affected TRN binding (50% reduction of AlphaScreen signal), whereas the R262A/R263A/K264A substitutions clearly mediated the largest effect (95% reduction in signal), although residual binding subsists (Fig. 5A). To control for correct folding of the interaction-defective mutant integrases (IN F185A/K186A/R187A/K188A and IN R262A/R263A/K264A ), we analyzed their interaction with LEDGF/p75 (32) and IN (Fig.  5B). LEDGF/p75 is a validated cofactor of integrase that interacts with integrase through its CCD domain (40 -42). The development of a robust AlphaScreen to measure interaction between LEDGF/p75 and IN has been described before (33). IN R262A/R263A/K264A bound LEDGF/p75 with a similar affinity as wild type IN (Fig. 5B) demonstrating that the overall structure of IN is not altered by these substitutions. Not surprisingly, IN F185A/K186A/R187A/K188A did not bind to LEDGF/p75 (Fig. 5B) because the substitutions K186A/R187A are known to inhibit tetramerization and LEDGF/p75 binding (43). To additionally control for the tertiary and quaternary structure, we tested the mutant integrases by size exclusion chromatography (Fig. 5C) (Fig. 5C). Because the F185A/ K186A/R187A/K188A substitutions clearly affect IN multimerization (Fig. 5C), we cannot exclude that the impact on TRN-SR2 binding is due to the altered multimerization equilibrium. Therefore and because the F185A/K186A/R187A/ K188A substitutions (located in the CCD) only modestly affected TRN-SR2 binding, we focused on the substitutions in C-terminal IN for the remainder of our study. Analysis of the Replication of HIV-1 Defective for Interaction with TRN-SR2-Because IN substitutions are known to have pleiotropic effects on HIV replication, we tried to find a set of mutations that only affects interaction with TRN-SR2. We screened different sets of double point mutants for their effect on binding to TRN-SR2 (Fig. 6A). Ultimately, we selected two double mutant INs (IN R263A/K264A and IN K266A/R269A ) with a 10-fold reduced affinity for TRN-SR2. We next assayed these two mutants for 3Ј-processing and overall integrase activity. IN R263A/K264A was defective (Ͻ25% of WT activity) for 3Ј-processing and strand transfer, whereas IN K266A/R269A could catalyze 3Ј-processing (Ͼ75% of WT activity) but not strand transfer (data not shown). As productive DNA binding is a prerequisite of catalytic activity, this assay indirectly reflects on the DNA binding capacity of IN K266A/R269A . Because IN K266A/R269A showed a strongly reduced affinity for TRN-SR2   -262-274). The fragment numbers refer to the positions of the amino acids in integrase and define the first and the last amino acid of each CTD fragment. Complexes were bound to glutathione donor beads and nickel-chelate acceptor beads. The signal, light emission, was measured using an EnVision Multilabel Reader (in counts/s). Each experiment was repeated three times. Representative graphs of single experiments are shown.

DISCUSSION
Recently, multiple research groups have reported on the importance of TRN-SR2 as a cellular cofactor of HIV replication (7, 18 -28). Although the exact role of TRN-SR2 in HIV replication is still under debate, there is a consensus that even moderate knockdown of this cofactor results in a strong inhibition of HIV replication at an early replication step prior to integration. Because TRN-SR2 belongs to the importin-␤ family of nuclear import factors (46 -49), it is not far-fetched to imagine a direct or indirect role in the nuclear import of the viral PIC (7,19,21). Recently, the hypothesis was formulated that TRN-SR2 also acts as a nuclear export factor for tRNA and HIV capsid (27), but this model awaits independent confirmation.
We identified TRN-SR2 as an interaction partner of HIV-1 IN in a yeast two-hybrid screen (19). Reverse yeast two-hybrid screening with TRN-SR2 only revealed IN as a viral binding partner. By employing Q-PCR and imaging of fluorescent viral particles, we could demonstrate that knockdown of TRN-SR2 blocks HIV replication at the step of nuclear import, a bottleneck for the infection of the host cell (19). Whether the physical interaction of TRN-SR2 with HIV-1 IN is the sole determinant for the nuclear import of the PIC and whether TRN-SR2 exerts other roles prior to and/or after nuclear import of the PIC still await further investigation (7, 18 -28). Here, we studied the molecular interaction between TRN-SR2 and HIV-1 IN to define the NLS in IN. Because IN has been proposed before to play a role in HIV nuclear import (5,6,11,12,50), several attempts have been made to define potential NLSs in IN. A bipartite NLS composed of two stretches ranging from amino acids 211 KELQKQITK 219 and 261 PRRKAK 266 (11) and a nonclassical NLS composed of amino acids 161 IIGQVRDQAEHLK 173 were described (31). Multiple IN mutations were inserted into viruses and tested for their impact on viral replication. IN mutants affecting HIV nuclear import were assayed for reduced a two-LTR circle formation (51,52). Despite these extensive studies, so far no single NLS was unambiguously identified as the determinant of HIV nuclear import. These older studies however were hampered by the lack of knowledge on the identity of the nuclear import factor.
Extended NLS sequences that result in the recognition of structural motifs in import factors are commonly found in cargo. To ensure a certain redundancy in cargo binding, it is unlikely that a single point mutation in the cargo will abrogate the interaction. With this in mind, we searched for the interaction domain in IN by assaying IN peptide stretches rather than using scanning or deletion mutagenesis. Mapping of the IN/TRN-SR2 interface was based on dissecting a large interaction domain into several small peptides and assaying those for their affinity for TRN-SR2. This method solved problems encountered with protein stability and solubility and allowed us to define INIP 1 and INIP 2 . Of note, the GST-peptide approach can be recommended for other protein-protein interaction studies when protein deletions are unstable or multipartite interaction regions occur.
Co-immunoprecipitation experiments demonstrated that TRN-SR2 interacts with both the catalytic core and the C-terminal domain of IN. Interaction studies with recombinant proteins confirmed the lack of firm contact with the NTD but revealed strong binding between TRN-SR2 and the CTD and weaker binding with the CCD prompting us to focus on these domains for further analysis.
In the original TRN-SR2 yeast two-hybrid paper, we described a virtual selected interaction domain based on the overlap between multiple clones that were picked up (19). The identified selected interaction domain covered the CCD, but a lot of the clones that where picked up also contained the CTD (19). As such, the identified clones further corroborate the present identification of a major interaction spot in the CTD and a weak second interaction spot in the CCD.
Here, we identified one stretch of amino acids in the CCD ( 170 EHLKTAVQMAVFIHNFKRKGGI 191 ) defined as the first integrase-derived interaction peptide (INIP 1 ) and two stretches of amino acids ( 214 QKQITKIQNFRVYYR 228 and 259 VVPRRKVKIIRDYGK 273 ) in the CTD. Of note, the peptides identified contain the potential NLS sequences that where previously identified (11, 31) but not confirmed due to a lack of an established interaction partner. By combining the two stretches of the CTD, we defined the second integrase-derived interaction peptide (INIP 2 ). By mutagenesis of INIP 1 and INIP 2 , we identified two stretches of amino acids that are crucial for the interaction. Phe-185/Lys-186/Arg-187/Lys-188 determine interaction in the CCD although Arg-262/ Arg-263/Lys-264 and Lys-266/Arg-269 are hot spots in the CTD. In the accompanying paper by Larue et al. (53), the role of the CTD residues Arg-262, Arg-263, and Lys-264 in binding to TRN-SR2 was independently confirmed.
After confirming their role in the CCD and the CTD, we  substitutions affect IN multimerization (Fig. 5C), we cannot exclude that the impact on TRN-SR2 binding is due to a modified multimerization equilibrium. Therefore and because the CCD substitutions only modestly affected TRN-SR2 binding, we focused on substitutions in the CTD for the remainder of our study. Because IN substitutions are known to have pleiotropic effects on HIV replication, we tried to find a set of mutations affecting only the interaction with TRN-SR2. We screened different sets of double point mutations for their binding to TRN-SR2. We selected two mutants (IN R263A/K264A and IN K266A/R269A ) with a strongly reduced affinity for TRN-SR2. Next, we assayed these two mutants for 3Ј-processing and overall integrase activity. Because IN K266A/R269A showed 10-fold reduced affinity for TRN-SR2 and retained 3Ј-processing activity, we selected this mutant for further virological studies. We introduced the mutations in a molecular clone and studied HIV replication. The K266A/R269A substitutions in IN rendered HIV replication-defective. However, due to a reverse transcription defect, we could not assess the impact of loss of IN/TRN-SR2 interaction on the nuclear import. The pleiotropic effects of IN mutations, especially in the CTD, are well known (51). First, the region between amino acids 220 and 270 of HIV IN plays a role in DNA binding (54). Furthermore, IN is a key protein within the viral particle that is not only responsible for integration catalysis but also interacts with other viral and cellular proteins to ensure integrity of the PIC. Within these nucleoprotein complexes, IN is known to interact with the reverse transcriptase. The interface of this interaction has been mapped to three crucial amino acids (K258A, W243E, and V250E) (55), but IN mutations outside of this interface (e.g. K186Q, R228A, R262A/R263A, R262A/K264A, K266A, and R269A) were shown to affect reverse transcription as well (51,52,56). Unfortunately, all mutants identified in this study are known to display reduced reverse transcription during replication (51,52,56), confounding the study of their role in the nuclear import of HIV in cellular replication assays. Still, insight into the hot spots that define the protein-protein interaction between IN and TRN-SR2 is necessary to guide future drug discovery efforts targeting the nuclear entry step of replication.