Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

Background

The formation of new infectious human immunodeficiency type 1 virus (HIV-1) mainly relies on the homo-multimerization of the viral structural polyprotein Pr55^Gag and on the recruitment of host factors. We have previously shown that the double-stranded RNA-binding protein Staufen 1 (Stau1), likely through an interaction between its third double-stranded RNA-binding domain (dsRBD3) and the nucleocapsid (NC) domain of Pr55^Gag, participates in HIV-1 assembly by influencing Pr55^Gag multimerization.

Results

We now report the fine mapping of Stau1/Pr55^Gag association using co-immunoprecipitation and live cell bioluminescence resonance energy transfer (BRET) assays. On the one hand, our results show that the Stau1-Pr55^Gag interaction requires the integrity of at least one of the two zinc fingers in the NC domain of Pr55^Gag but not that of the NC N-terminal basic region. Disruption of both zinc fingers dramatically impeded Pr55^Gag multimerization and virus particle release. In parallel, we tested several Stau1 deletion mutants for their capacity to influence Pr55^Gag multimerization using the Pr55^Gag/Pr55^Gag BRET assay in live cells. Our results revealed that a molecular determinant of 12 amino acids at the N-terminal end of Stau1 is necessary to increase Pr55^Gag multimerization and particle release. However, this region is not required for Stau1 interaction with the viral polyprotein Pr55^Gag.

Conclusion

These data highlight that Stau1 is a modular protein and that Stau1 influences Pr55^Gag multimerization via 1) an interaction between its dsRBD3 and Pr55^Gag zinc fingers and 2) a regulatory domain within the N-terminus that could recruit host machineries that are critical for the completion of new HIV-1 capsids.     

Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

The human immunodeficiency virus type 1 (HIV-1) Pr55^gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55^gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55^gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55^gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55^gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55^gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55^gag and p17MA in virions and membranes.     

Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

The precursor group-specific antigen (pr55^Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55^Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55^Gag expression, the synthesis of other HIV-1 proteins, including that of pr160^Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55^Gag for virus assembly and production.     

The I Domain Is Required for Efficient Plasma Membrane Binding of Human Immunodeficiency Virus Type 1 Pr55Gag

The interaction of the human immunodeficiency virus type 1 (HIV-1) Pr55^Gag molecule with the plasma membrane of an infected cell is an essential step of the viral life cycle. Myristic acid and positively charged residues within the N-terminal portion of MA constitute the membrane-binding domain of Pr55^Gag. A separate assembly domain, termed the interaction (I) domain, is located nearer the C-terminal end of the molecule. The I domain is required for production of dense retroviral particles, but has not previously been described to influence the efficiency of membrane binding or the subcellular distribution of Gag. This study used a series of Gag-green fluorescent protein fusion constructs to define a region outside of MA which determines efficient plasma membrane interaction. This function was mapped to the nucleocapsid (NC) region of Gag. The minimal region in a series of C-terminally truncated Gag proteins conferring plasma membrane fluorescence was identified as the N-terminal 14 amino acids of NC. This same region was sufficient to create a density shift in released retrovirus-like particles from 1.13 to 1.17 g/ml. The functional assembly domain previously termed the I domain is thus required for the efficient plasma membrane binding of Gag, in addition to its role in determining the density of released particles. We propose a model in which the I domain facilitates the interaction of the N-terminal membrane-binding domain of Pr55^Gag with the plasma membrane.     

An electron spin resonance study of the novel radical cation produced during the horseradish peroxidase-catalyzed oxidation of tetramethylhydrazine

Thrombin stimulation of [³²P]-prelabeled platelets induces a rapid decrease of the radioactivity from phosphatidylinositol-4,5-bisphosphate. No significant change is observed in phosphatidylinositol-4-monophosphate. The initial, thrombin-induced decrease of phosphatidylinositol-4,5-bisphosphate is not inhibited by cytochalasin D or by compounds that interfere with the mobilization of Ca²⁺ such as 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the calmodulin-antagonist, trifluoperazine, prostacyclin and cyclic AMP. Our information indicates that the rapid loss of phosphatidylinositol-4,5-bisphosphate is linked to receptor activation and insensitive to Ca²⁺-mobilization.     

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77^Gag along with in cell gRNA packaging study identified two Pr77^Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77^Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.     

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

Certain immune-driven mutations in HIV-1, such as those arising in p24^Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24^Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24^Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.     

Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6<Superscript>Gag</Superscript>

Background

HIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6^Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6^Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6^Gag had only minor effects.

Findings

We introduced mutations into p6^Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.

Conclusions

An HIV-1 variant carrying a conservative S40N exchange in p6^Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

     

Inositol-containing lipids in suspension-cultured plant cells: an isotopic study

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [³²P]orthosphosphate, [2-³H]glycerol, and myo-[2-³H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [³²P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.     

Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.     

Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

The p6^Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6^Gag between Y³⁶ and P³⁷, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6^Gag proteins in HIV-1_MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6^Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6^Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41^TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6^Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.     

Ubiquitin Is Covalently Attached to the p6Gag Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6^Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12^Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6^Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.     

Novel cytotoxic T-lymphocyte escape mutation by a three-amino-acid insertion in the human immunodeficiency virus type 1 p6Pol and p6Gag late domain associated with drug resistance

Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8⁺ CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6^Pol protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6^Pol epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6^Gag late domain, PTAPP(APP). Interestingly, this p6^Pol insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6^Gag late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.     

Resveratrol inhibits polyphosphoinositide metabolism in activated platelets

The effects of resveratrol (trans-3,4′,5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P₂), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P₃) were monitored in blood platelets prelabelled with [³²P]P_i. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of ³²P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P₂ available for signalling in these cells.     

Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting

During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P₂ analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P₂ localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P₂-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.     

Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P₂ in Ca²⁺ homeostasis and excitation–contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein–tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca²⁺ transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca²⁺ release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP–expressing fibers challenged by 5-s-long depolarizing pulses, the Ca²⁺ level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP–expressing fibers showed a reversible depression of Ca²⁺ release during trains, with the peak Ca²⁺ transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP–expressing fibers. Cav1.1 Ca²⁺ channel–mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ₁PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule–bound PLCδ₁PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P₂ level is tightly maintained in the transverse tubule membrane of the muscle fibers, and that VSP-induced depletion of PtdIns(4,5)P₂ impairs voltage-activated Ca²⁺ release from the SR. Because Ca²⁺ release is thought to be independent from InsP₃ signaling, the effect likely results from an interaction between PtdIns(4,5)P₂ and a protein partner of the E-C coupling machinery.     

HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA^Lys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA^Lys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA^Lys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Unexpectedly, the affinity of MA for tRNA^Lys3 (K_d = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P₂-enriched liposomes (K_d(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA^Lys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P₂, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA^Lys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.