Molecular Determinants of PQBP1 Binding to the HIV-1 Capsid Lattice.

Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1's primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding.

Structural basis of HIV-1 maturation inhibitor binding and activity.

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

Purification and structure of luminal domain C of human Niemann-Pick C1 protein.

Niemann-Pick C1 protein (NPC1) is a membrane protein that primarily resides in late endosomes and lysosomes, and plays an important role in cholesterol homeostasis in the cell. The second luminal domain of NPC1 (NPC1-C) serves as the intracellular receptor for Ebola and Marburg viruses. Here, the recombinant production of nonglycosylated and glycosylated NPC1-C and a new crystal form of the nonglycosylated protein are reported. The crystals belonged to space group P21 and diffracted to 2.3 Šresolution. The structure is similar to other reported structures of NPC1-C, with differences observed in the protruding loops when compared with NPC1-C in complex with Ebola virus glycoprotein or NPC2.

A molecular switch modulates assembly and host factor binding of the HIV-1 capsid.

The HIV-1 capsid is a fullerene cone made of quasi-equivalent hexamers and pentamers of the viral CA protein. Typically, quasi-equivalent assembly of viral capsid subunits is controlled by a molecular switch. Here, we identify a Thr-Val-Gly-Gly motif that modulates CA hexamer/pentamer switching by folding into a 310 helix in the pentamer and random coil in the hexamer. Manipulating the coil/helix configuration of the motif allowed us to control pentamer and hexamer formation in a predictable manner, thus proving its function as a molecular switch. Importantly, the switch also remodels the common binding site for host factors that are critical for viral replication and the new ultra-potent HIV-1 inhibitor lenacapavir. This study reveals that a critical assembly element also modulates the post-assembly and viral replication functions of the HIV-1 capsid and provides new insights on capsid function and inhibition.

Strain and rupture of HIV-1 capsids during uncoating.

SignificanceThe mature capsids of HIV-1 are transiently stable complexes that self-assemble around the viral genome during maturation, and uncoat to release preintegration complexes that archive a double-stranded DNA copy of the virus in the host cell genome. However, a detailed view of how HIV cores rupture remains lacking. Here, we elucidate the physical properties involved in capsid rupture using a combination of large-scale all-atom molecular dynamics simulations and cryo-electron tomography. We find that intrinsic strain on the capsid forms highly correlated patterns along the capsid surface, along which cracks propagate. Capsid rigidity also increases with high strain. Our findings provide fundamental insight into viral capsid uncoating.

Poly(ADP-ribose) potentiates ZAP antiviral activity.

Zinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of an adenosine diphosphate-containing unit of PAR. Mutation of the PAR binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus and murine leukemia virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.

Structural evidence that MOAP1 and PEG10 are derived from retrovirus/retrotransposon Gag proteins.

The Gag proteins of retroviruses play an essential role in virus particle assembly by forming a protein shell or capsid and thus generating the virion compartment. A variety of human proteins have now been identified with structural similarity to one or more of the major Gag domains. These human proteins are thought to have been evolved or "domesticated" from ancient integrations due to retroviral infections or retrotransposons. Here, we report that X-ray crystal structures of stably folded domains of MOAP1 (modulator of apoptosis 1) and PEG10 (paternally expressed gene 10) are highly similar to the C-terminal capsid (CA) domains of cognate Gag proteins. The structures confirm classification of MOAP1 and PEG10 as domesticated Gags, and suggest that these proteins may have preserved some of the key interactions that facilitated assembly of their ancestral Gags into capsids.

Determination of Histidine Protonation States in Proteins by Fast Magic Angle Spinning NMR.

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40-62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)-based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.

Structural Determinants of Virion Assembly and Release in the C Terminus of the Mason-Pfizer Monkey Virus Capsid Protein.

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo. The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro.

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract-dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy.

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

TRIM5α self-assembly and compartmentalization of the HIV-1 viral capsid.

The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α.

Biochemical Reconstitution of HIV-1 Assembly and Maturation.

The assembly of an orthoretrovirus such as HIV-1 requires the coordinated functioning of multiple biochemical activities of the viral Gag protein. These activities include membrane targeting, lattice formation, packaging of the RNA genome, and recruitment of cellular cofactors that modulate assembly. In most previous studies, these Gag activities have been investigated individually, which provided somewhat limited insight into how they functionally integrate during the assembly process. Here, we report the development of a biochemical reconstitution system that allowed us to investigate how Gag lattice formation, RNA binding, and the assembly cofactor inositol hexakisphosphate (IP6) synergize to generate immature virus particles in vitro The results identify an important rate-limiting step in assembly and reveal new insights into how RNA and IP6 promote immature Gag lattice formation. The immature virus-like particles can be converted into mature capsid-like particles by the simple addition of viral protease, suggesting that it is possible in principle to fully biochemically reconstitute the sequential processes of HIV-1 assembly and maturation from purified components.IMPORTANCE Assembly and maturation are essential steps in the replication of orthoretroviruses such as HIV-1 and are proven therapeutic targets. These processes require the coordinated functioning of the viral Gag protein's multiple biochemical activities. We describe here the development of an experimental system that allows an integrative analysis of how Gag's multiple functionalities cooperate to generate a retrovirus particle. Our current studies help to illuminate how Gag synergizes the formation of the virus compartment with RNA binding and how these activities are modulated by the small molecule IP6. Further development and use of this system should lead to a more comprehensive understanding of the molecular mechanisms of HIV-1 assembly and maturation and may provide new insights for the development of antiretroviral drugs.

Hierarchical assembly governs TRIM5α recognition of HIV-1 and retroviral capsids.

TRIM5α is a restriction factor that senses incoming retrovirus cores through an unprecedented mechanism of nonself recognition. TRIM5α assembles a hexagonal lattice that avidly binds the capsid shell, which surrounds and protects the virus core. The extent to which the TRIM lattice can cover the capsid and how TRIM5α directly contacts the capsid surface have not been established. Here, we apply cryo-electron tomography and subtomogram averaging to determine structures of TRIM5α bound to recombinant HIV-1 capsid assemblies. Our data support a mechanism of hierarchical assembly, in which a limited number of basal interaction modes are successively organized in increasingly higher-order structures that culminate in a TRIM5α cage surrounding a retroviral capsid. We further propose that cage formation explains the mechanism of restriction and provides the structural context that links capsid recognition to ubiquitin-dependent processes that disable the retrovirus.

Restriction of HIV-1 and other retroviruses by TRIM5.

Mammalian cells express a variety of innate immune proteins - known as restriction factors - which defend against invading retroviruses such as HIV-1. Two members of the tripartite motif protein family - TRIM5α and TRIMCyp - were identified in 2004 as restriction factors that recognize and inactivate the capsid shell that surrounds and protects the incoming retroviral core. Research on these TRIM5 proteins has uncovered a novel mode of non-self recognition that protects against cross-species transmission of retroviruses. Our developing understanding of the mechanism of TRIM5 restriction underscores the concept that core uncoating and reverse transcription of the viral genome are coordinated processes rather than discrete steps of the post-entry pathway of retrovirus replication. In this Review, we provide an overview of the current state of knowledge of the molecular mechanism of TRIM5-mediated restriction, highlight recent advances and discuss implications for the development of capsid-targeted antiviral therapeutics.

Maturation of retroviruses.

During retrovirus maturation, cleavage of the precursor structural Gag polyprotein by the viral protease induces architectural rearrangement of the virus particle from an immature into a mature, infectious form. The structural rearrangement encapsidates the viral RNA genome in a fullerene capsid, producing a diffusible viral core that can initiate infection upon entry into the cytoplasm of a host cell. Maturation is an important therapeutic window against HIV-1. In this review, we highlight recent breakthroughs in understanding of the structures of retroviral immature and mature capsid lattices that define the boundary conditions of maturation and provide novel insights on capsid transformation. We also discuss emerging insights on encapsidation of the viral genome in the mature capsid, as well as remaining questions for further study.

Author Correction: Inositol phosphates are assembly co-factors for HIV-1.

In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as '6BH5, 6BHT and 6BHS' instead of '6BHR, 6BHT and 6BHS'; this has been corrected online.

Inositol phosphates are assembly co-factors for HIV-1.

A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.

Nup153 Unlocks the Nuclear Pore Complex for HIV-1 Nuclear Translocation in Nondividing Cells.

Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.IMPORTANCE One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.

General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 µM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.