NMR, biophysical, and biochemical studies reveal the minimal Calmodulin binding domain of the HIV-1 matrix protein

Subcellular distribution of Calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM has been shown to interact and co-localize with the HIV-1 Gag protein in infected cells. However, the precise molecular mechanism of this interaction is not known. Binding of Gag to CaM is dependent on calcium and is mediated by the N-terminal-myristoylated matrix (myr(+)MA) domain. We have recently shown that CaM binding induces a conformational change in the MA protein, triggering exposure of the myristate group. To unravel the molecular mechanism of CaM-MA interaction and to identify the minimal CaM binding domain of MA, we devised multiple approaches utilizing NMR, biochemical, and biophysical methods. Short peptides derived from the MA protein have been examined. Our data revealed that whereas peptides spanning residues 11-28 (MA-(11-28)) and 31-46 (MA-(31-46)) appear to bind preferentially to the C-terminal lobe of CaM, a peptide comprising residues 11-46 (MA-(11-46)) appears to engage both domains of CaM. Limited proteolysis data conducted on the MA-CaM complex yielded a MA peptide (residues 8-43) that is protected by CaM and resistant to proteolysis. MA-(8-43) binds to CaM with a very high affinity (dissociation constant = 25 nm) and in a manner that is similar to that observed for the full-length MA protein. The present findings provide new insights on how MA interacts with CaM that may ultimately help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle.     

Binding of calmodulin to the HIV-1 matrix protein triggers myristate exposure

Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca(2+)-binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(+)MA) domain. Herein, we demonstrate that CaM binds to myr(+)MA with a dissociation constant (K(d)) of ～2 μm and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(+)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.     

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.     

Binding of the Human Immunodeficiency Virus Type 1 Gag Protein to the Viral RNA Encapsidation Signal in the Yeast Three-Hybrid System

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496–8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVΨ). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVΨ signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.     

Do calmodulin binding IQ motifs have built‐in capping domains?

Most calmodulin (CaM) targets are α‐helices. It is not clear if CaM induces the adoption of an α‐helix configuration to its targets or if those targets are selected as they spontaneously adopt an α‐helical conformation. Other than an α‐helix propensity, there is a great variety of CaM targets with little more in common. One exception to this rule is the IQ site that can be recognized in a number of targets, such as those ion channels belonging to the KCNQ family. Although there is negligible sequence similarity between the IQ motif and the docking site on SK2 channels, both adopt a similar three‐dimensional disposition. The isolated SK2 target presents a pre‐folded core region that becomes fully α‐helical upon binding to CaM. The existence of this pre‐folded state suggests the occurrence of capping within CaM targets. In this review, we examine the capping properties within the residues flanking this core domain, and relate known IQ motifs and capping.     

Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

Old world monkey TRIM5α is a host factor that restricts human immunodeficiency virus type-1 (HIV-1) infection. Previously, we reported that rhesus macaque TRIM5α (RhTRIM5α) restricts HIV-1 production by inducing degradation of precursor Gag. Since suppressor of cytokine signaling 1 (SOCS1) is known to enhance HIV-1 production by rescuing Gag from lysosomal degradation, we examined if SOCS1 is involved in RhTRIM5α-mediated late restriction. Over-expression of SOCS1 restored HIV-1 production in the presence of RhTRIM5α to a level comparable to that in the absence of RhTRIM5α in terms of titer and viral protein expression. Co-immunoprecipitation studies revealed that SOCS1 physically interacted with RhTRIM5α. Over-expression of SOCS1 affected RhTRIM5α expression in a dose-dependent manner, which was not reversed by proteasome inhibitors. In addition, SOCS1 and RhTRIM5α were detected in virus-like particles. These results suggest that SOCS1 alleviates RhTRIM5α-mediated regulation in the late phase of HIV-1 life cycle probably due to the destabilization of RhTRIM5α.     

Domain Swapping and Different Oligomeric States for the Complex Between Calmodulin and the Calmodulin-Binding Domain of Calcineurin A

Background

Calmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A.

Methodology/Principal Findings

We have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel ‘1-11’ type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases.

Conclusions/Significance

Our results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1∶1 stoichiometry, in addition to the previously observed 2∶2 arrangement in the crystal state. At the structural level, going from 2∶2 association to two 1∶1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM.     

The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.     

N-Terminal and C-Terminal Domains of Calmodulin Mediate FADD and TRADD Interaction

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.     

Defective HIV-1 Particle Assembly in AP-3-Deficient Cells Derived from Patients with Hermansky-Pudlak Syndrome Type 2

Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the β3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type β3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.     

Crystal Structures of Human CaMKIα Reveal Insights into the Regulation Mechanism of CaMKI

Human calcium/calmodulin-dependent protein kinase I (CaMKI) plays pivotal roles in the nervous system. The activity of human CaMKI is regulated by a regulatory region including an autoinhibitory segment and a CaM-binding segment. We report here four structures of three CaMKIα truncates in apo form and in complexes with ATP. In an apo, autoinhibited structure, the activation segment adopts a unique helical conformation which together with the autoinhibitory segment constrains helices αC and αD in inactive conformations, sequesters Thr177 from being phosphorylated, and occludes the substrate-binding site. In an ATP-bound, inactive structure, the activation segment is largely disordered and the CaM-binding segment protrudes out ready for CaM binding. In an ATP-bound, active structure, the regulatory region is dissociated from the catalytic core and the catalytic site assumes an active conformation. Detailed structural analyses reveal the interplay of the regulatory region, the activation segment, and the nucleotide-binding site in the regulation of CaMKI.     

The NC2 Domain of Type IX Collagen Determines the Chain Register of the Triple Helix

Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35–50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)₃ or (α2)₃. For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)₃ as the strongest binding construct, whereas the homo-trimer of (α2)₃ showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.     

Identification of the Calmodulin-Binding Domains of Fas Death Receptor

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas–mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209–239 (Fas-Pep1) and 251–288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD–CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.     

Cryptic Determinant of α4β7 Binding in the V2 Loop of HIV-1 gp120

The peptide segment of the second variable loop of HIV-1 spanning positions 166–181 harbors two functionally important sites. The first, spanning positions 179–181, engages the human α4β7 integrin receptor which is involved in T-cell gut-homing and may play a role in human immunodeficiency virus (HIV)-host cell interactions. The second, at positions 166–178, is a major target of anti-V2 antibodies elicited by the ALVAC/AIDSVAX vaccine used in the RV144 clinical trial. Notably, these two sites are directly adjacent, but do not overlap. Here, we report the identity of a second determinant of α4β7 binding located at positions 170–172 of the V2 loop. This segment – tripeptide QRV^170–172– is located within the second site, yet functionally affects the first site. The absence of this segment abrogates α4β7 binding in peptides bearing the same sequence from position 173–185 as the V2 loops of the RV144 vaccines. However, peptides exhibiting V2 loop sequences from heterologous HIV-1 strains that include this QRV^170–172 motif bind the α4β7 receptor on cells. Therefore, the peptide segment at positions 166–178 of the V2 loop of HIV-1 viruses appears to harbor a cryptic determinant of α4β7 binding. Prior studies show that the anti-V2 antibody response elicited by the RV144 vaccine, along with immune pressure inferred from a sieve analysis, is directed to this same region of the V2 loop. Accordingly, the anti-V2 antibodies that apparently reduced the risk of infection in the RV144 trial may have functioned by blocking α4β7-mediated HIV-host cell interactions via this cryptic determinant.     

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.     

Ionic interactions are essential for TRPV1 C-terminus binding to calmodulin

Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the C-terminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT’s binding to CaM, with arginine R785 being the most essential residue. The homology modelling suggests that a CaM-binding motif of TRPV1-CT forms an alpha helix that docks into the central cavity of CaM.     

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein,X-ray cross-complementing group 1, with DNA polymerase beta

Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase β (β-Pol). Here we report a site- directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to β-Pol. Mutant proteins defective in XRCC1-NTD interaction with β-Pol and with a β-Pol–gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass β-strand D and E of the five-stranded β-sheet (βABGDE) and the protruding α2 helix of the XRCC1-NTD. Mutations that included F67A (βD), E69K (βD), V86R (βE) on the five-stranded β-sheet and deletion of the α2 helix, but not mutations within α2, abolished binding of the XRCC1-NTD to β-Pol. A Y136A mutant abolished β-Pol binding, and a R109S mutant reduced β-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and δ-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the α2 helix and the βD and βE strands of XRCC1-NTD to the energetics of β-Pol binding. Establishing the direct contacts in the β-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.     

Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P(2) interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P(2) dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P(2) is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P(2). Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P(2). Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P(2) and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.