Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.

Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

HTLV-1 is an oncogenic human retrovirus and the etiologic agent of the highly aggressive ATL malignancy. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in the pathogenic process. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral lifecycle and directly contributes to its pathogenic potential. Herein, we identified the cellular transcription factor YBX1 as a binding partner for HBZ. We found YBX1 activated transcription and enhanced Tax-mediated transcription from the viral 5' LTR promoter. Interestingly, YBX1 also interacted with Tax. shRNA-mediated loss of YBX1 decreased transcript and protein abundance of both Tax and HBZ in HTLV-1-transformed T-cell lines, as well as Tax association with the 5' LTR. Conversely, YBX1 transcriptional activation of the 5' LTR promoter was increased in the absence of HBZ. YBX1 was found to be associated with both the 5' and 3' LTRs in HTLV-1-transformed and ATL-derived T-cell lines. Together, these data suggest that YBX1 positively influences transcription from both the 5' and 3' promoter elements. YBX1 is able to interact with Tax and help recruit Tax to the 5' LTR. However, through interactions with HBZ, YBX1 transcriptional activation of the 5' LTR is repressed.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

The Drug-Induced Interface That Drives HIV-1 Integrase Hypermultimerization and Loss of Function.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target. IMPORTANCE Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.

Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection.

Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.

Structural and Mechanistic Bases of Viral Resistance to HIV-1 Capsid Inhibitor Lenacapavir.

Lenacapavir (LEN) is a long-acting, highly potent HIV-1 capsid (CA) inhibitor. The evolution of viral variants under the genetic pressure of LEN identified Q67H, N74D, and Q67H/N74D CA substitutions as the main resistance associated mutations (RAMs). Here, we determined high-resolution structures of CA hexamers containing these RAMs in the absence and presence of LEN. Our findings reveal that the Q67H change induces a conformational switch, which adversely affects the inhibitor binding. In the unliganded protein, the His67 side chain adopts the closed conformation by projecting into the inhibitor binding pocket and thereby creating steric hindrance with respect to LEN. Upon the inhibitor binding, the His67 side chain repositions to the open conformation that closely resembles the Gln67 side chain in the WT protein. We propose that the switch from the closed conformation to the open conformation, which is needed to accommodate LEN, accounts for the reduced inhibitor potency with respect to the Q67H CA variant. The N74D CA change results in the loss of a direct hydrogen bond and in induced electrostatic repulsions between CA and LEN. The double Q67H/N74D substitutions exhibited cumulative effects of respective single amino acid changes. An examination of LEN binding kinetics to CA hexamers revealed that Q67H and N74D CA changes adversely influenced the inhibitor binding affinity (KD) by primarily affecting the dissociation rate constant (koff). We used these structural and mechanistic findings to rationally modify LEN. The resulting analog exhibited increased potency against the Q67H/N74D viral variant. Thus, our studies provide a means for the development of second-generation inhibitors with enhanced barriers to resistance. IMPORTANCE LEN is an investigational long-acting agent for future HIV-1 treatment regimens. While ongoing clinical trials have highlighted a largely beneficial profile of LEN for the treatment of HIV-1 infected people with limited therapy options, one notable shortcoming is a relatively low barrier of viral resistance to the inhibitor. Cell culture-based viral breakthrough assays identified N74D, Q67H, and N74D/Q67H capsid changes as the main resistance associated mutations (RAMs). N74D and Q67H capsid substitutions have also emerged in clinical trials in some patients who received subcutaneous LEN. Understanding the structural basis behind viral resistance to LEN is expected to aid in the rational development of improved inhibitors with enhanced barriers to resistance. Here, we report high resolution structures of the main drug resistant capsid variants, which provide mechanistic insight into the viral resistance to LEN. We used these findings to develop an improved inhibitor, which exhibited enhanced activity against the viral Q67H/N74D capsid phenotype compared with that of parental LEN.

HIV-1 Preintegration Complex Preferentially Integrates the Viral DNA into Nucleosomes Containing Trimethylated Histone 3-Lysine 36 Modification and Flanking Linker DNA.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA.

HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells. Here we show that compensatory substitutions in nearby negatively charged aspartic acid residues (i.e., D256N, D270N) restore the ability of IN to bind gRNA for these mutant viruses and result in the formation of accurately matured infectious virions. Similarly, individual charge reversal substitutions at D256 as well as other nearby positions (i.e., D278, D279) are all sufficient to enable the respective IN mutants to bind gRNA, and subsequently restore reverse transcription and virion infectivity. Taken together, our findings reveal the importance of highly pliable electrostatic interactions in IN-gRNA binding.

Multimodal Functionalities of HIV-1 Integrase.

Integrase is the retroviral protein responsible for integrating reverse transcripts into cellular genomes. Co-packaged with viral RNA and reverse transcriptase into capsid-encased viral cores, human immunodeficiency virus 1 (HIV-1) integrase has long been implicated in reverse transcription and virion maturation. However, the underlying mechanisms of integrase in these non-catalytic-related viral replication steps have remained elusive. Recent results have shown that integrase binds genomic RNA in virions, and that mutational or pharmacological disruption of integrase-RNA binding yields eccentric virion particles with ribonucleoprotein complexes situated outside of the capsid shell. Such viruses are defective for reverse transcription due to preferential loss of integrase and viral RNA from infected target cells. Parallel research has revealed defective integrase-RNA binding and eccentric particle formation as common features of class II integrase mutant viruses, a phenotypic grouping of viruses that display defects at steps beyond integration. In light of these new findings, we propose three new subclasses of class II mutant viruses (a, b, and c), all of which are defective for integrase-RNA binding and particle morphogenesis, but differ based on distinct underlying mechanisms exhibited by the associated integrase mutant proteins. We also assess how these findings inform the role of integrase in HIV-1 particle maturation.

Identification and Optimization of a Novel HIV-1 Integrase Inhibitor.

Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1, like all retroviruses, stably integrates its vDNA copy into host chromatin, a process allowing for permanent infection. This essential step for HIV-1 replication is catalyzed by viral integrase (IN) and aided by cellular protein LEDGF/p75. In addition, IN is also crucial for proper virion maturation as it interacts with the viral RNA genome to ensure encapsulation of ribonucleoprotein complexes within the protective capsid core. These key functions make IN an attractive target for the development of inhibitors with various mechanisms of action. We conducted a high-throughput screen (HTS) of ∼370,000 compounds using a homogeneous time-resolved fluorescence-based assay capable of capturing diverse inhibitors targeting multifunctional IN. Our approach revealed chemical scaffolds containing diketo acid moieties similar to IN strand transfer inhibitors (INSTIs) as well as novel compounds distinct from all current IN inhibitors including INSTIs and allosteric integrase inhibitors (ALLINIs). Specifically, our HTS resulted in the discovery of compound 12, with a novel IN inhibitor scaffold amenable for chemical modification. Its more potent derivative 14e similarly inhibited catalytic activities of WT and mutant INs containing archetypical INSTI- and ALLINI-derived resistant substitutions. Further SAR-based optimization resulted in compound 22 with an antiviral EC50 of ∼58 µM and a selectivity index of >8500. Thus, our studies identified a novel small-molecule scaffold for inhibiting HIV-1 IN, which provides a promising platform for future development of potent antiviral agents to complement current HIV-1 therapies.

HIV-1 integrase binding to genomic RNA 5'-UTR induces local structural changes in vitro and in virio.

BACKGROUND: During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. HIV-1 integrase (IN) binds the viral genomic RNA (gRNA) and impairment of IN-gRNA binding leads to mis-localization of the nucleocapsid protein (NC)-condensed viral ribonucleoprotein complex outside the capsid core. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; however, the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood. RESULTS: Using crosslinking-coupled selective 2'-hydroxyl acylation analyzed by primer extension (XL-SHAPE), we characterized the interaction of IN and NC with the HIV-1 gRNA 5'-untranslated region (5'-UTR). NC preferentially bound to the packaging signal (Psi) and a UG-rich region in U5, irrespective of the presence of IN. IN alone also bound to Psi but pre-incubation with NC largely abolished this interaction. In contrast, IN specifically bound to and affected the nucleotide (nt) dynamics of the apical loop of the transactivation response element (TAR) and the polyA hairpin even in the presence of NC. SHAPE probing of the 5'-UTR RNA in virions produced from allosteric IN inhibitor (ALLINI)-treated cells revealed that while the global secondary structure of the 5'-UTR remained unaltered, the inhibitor treatment induced local reactivity differences, including changes in the apical loop of TAR that are consistent with the in vitro results. CONCLUSIONS: Overall, the binding interactions of NC and IN with the 5'-UTR are largely orthogonal in vitro. This study, together with previous probing experiments, suggests that IN and NC binding in vitro and in virio lead to only local structural changes in the regions of the 5'-UTR probed here. Accordingly, disruption of IN-gRNA binding by ALLINI treatment results in local rather than global secondary structure changes of the 5'-UTR in eccentric virus particles.

A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site.

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

ATR prevents Ca2+ overload-induced necrotic cell death through phosphorylation-mediated inactivation of PARP1 without DNA damage signaling.

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+ /ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling.

Sec24C is an HIV-1 host dependency factor crucial for virus replication.

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus-host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and identified Sec24C as an HIV-1 host dependency factor. Gene deletion and complementation in Jurkat cells revealed that Sec24C facilitates infection and markedly enhances HIV-1 spreading infection. Downregulation of Sec24C in HeLa cells substantially reduced HIV-1 core stability and adversely affected reverse transcription, nuclear import and infectivity. Live-cell microscopy showed that Sec24C co-trafficked with HIV-1 cores in the cytoplasm during virus ingress. Biochemical assays demonstrated that Sec24C directly and specifically interacted with hexameric capsid lattices. A 2.3-Å resolution crystal structure of Sec24C228-242 in the complex with a capsid hexamer revealed that the Sec24C FG-motif bound to a pocket comprised of two adjoining capsid subunits. Combined with previous data1-4, our findings indicate that a capsid-binding FG-motif is conserved in unrelated proteins present in the cytoplasm (Sec24C), the nuclear pore (Nup153; refs. 3,4) and the nucleus (CPSF6; refs. 1,2). We propose that these virus-host interactions during HIV-1 trafficking across different cellular compartments are crucial for productive infection of target cells.

Structural and mechanistic bases for a potent HIV-1 capsid inhibitor.

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis.

A large number of human immunodeficiency virus 1 (HIV-1) integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

Exploring the Free-Energy Landscape and Thermodynamics of Protein-Protein Association.

We report the free-energy landscape and thermodynamics of the protein-protein association responsible for the drug-induced multimerization of HIV-1 integrase (IN). Allosteric HIV-1 integrase inhibitors promote aberrant IN multimerization by bridging IN-IN intermolecular interactions. However, the thermodynamic driving forces and kinetics of the multimerization remain largely unknown. Here, we explore the early steps in the IN multimerization by using umbrella sampling and unbiased molecular dynamics simulations in explicit solvent. In direct simulations, the two initially separated dimers spontaneously associate to form near-native complexes that resemble the crystal structure of the aberrant tetramer. Most strikingly, the effective interaction of the protein-protein association is very short-ranged: the two dimers associate rapidly within tens of nanoseconds when their binding surfaces are separated by d ≤ 4.3 Å (less than two water diameters). Beyond this distance, the oligomerization kinetics appears to be diffusion controlled with a much longer association time. The free-energy profile also captured the crucial role of allosteric IN inhibitors in promoting multimerization and explained why several C-terminal domain mutations are remarkably resistant to the drug-induced multimerization. The results also show that at small separation, the protein-protein binding process contains two consecutive phases with distinct thermodynamic signatures. First, interprotein water molecules are expelled to the bulk, resulting in a small increase in entropy, as the solvent entropy gain from the water release is nearly cancelled by the loss of side-chain entropies as the two proteins approach each other. At shorter distances, the two dry binding surfaces adapt to each other to optimize their interaction energy at the expense of further protein configurational entropy loss. Although the binding interfaces feature clusters of hydrophobic residues, overall, the protein-protein association in this system is driven by enthalpy and opposed by entropy.