Biomedical subjects

Wesley I Sundquist

Publications and source records attributed to Wesley I Sundquist.

31 records · Page 2Linked to original sources

PubMed2003

The protein network of HIV budding.

HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.

Animals↗

PubMed2003

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein.

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

Cell Line↗

PubMed2003

Good to CU.

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APOBEC-3G Deaminase↗

PubMed2003

Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly.

Although retroviruses from different genera form morphologically distinct capsids, we have proposed that all of these structures are composed of similar hexameric arrays of capsid (CA) protein subunits and that their distinct morphologies reflect different distributions of pentameric declinations that allow the structures to close. Consistent with this model, CA proteins from both HIV-1 and Rous sarcoma virus (RSV) form similar hexagonal lattices. However, recent structural studies have suggested that the Moloney murine leukemia virus (M-MuLV) CA protein may assemble differently. We now report an independent three-dimensional reconstruction of two-dimensional crystals of M-MuLV CA. This new reconstruction reveals a hexameric lattice that is similar to those formed by HIV-1 and RSV CA, supporting a generalized model for retroviral capsid assembly.

Animals↗

PubMed2003

Structure and ubiquitin binding of the ubiquitin-interacting motif.

Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4.

Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel beta-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the "Ile-44" surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin.

Animals↗

PubMed2003

Structural insights into the catalytic mechanism of cyclophilin A.

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.

Amino Acid Sequence↗

PubMed2003

Functional surfaces of the human immunodeficiency virus type 1 capsid protein.

The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.

Amino Acid Sequence↗

PubMed2002

Structure and functional interactions of the Tsg101 UEV domain.

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.

Amino Acid Sequence↗

PubMed2002

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A.

Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89-Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CA(N)). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CA(N) has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CA(N) but also catalyzes efficiently the cis/trans isomerization of the Gly-89-Pro-90 peptide bond. In addition, conformational changes in CA(N) distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.

Capsid↗

PubMed2002

Mechanisms of enveloped RNA virus budding.

To spread infection, enveloped viruses must bud from infected host cells. Recent research indicates that HIV and other enveloped RNA viruses bud by appropriating the cellular machinery that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies. This new model of virus budding has many potential implications for cell biology and viral pathogenesis.

Cell Membrane↗

PubMed2002

Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein.

The structural proteins of HIV and Ebola display PTAP peptide motifs (termed 'late domains') that recruit the human protein Tsg101 to facilitate virus budding. Here we present the solution structure of the UEV (ubiquitin E2 variant) binding domain of Tsg101 in complex with a PTAP peptide that spans the late domain of HIV-1 p6(Gag). The UEV domain of Tsg101 resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. Each PTAP residue makes important contacts, and the Ala 9-Pro 10 dipeptide binds in a deep pocket of the UEV domain that resembles the X-Pro binding pockets of SH3 and WW domains. The structure reveals the molecular basis of HIV PTAP late domain function and represents an attractive starting point for the design of novel inhibitors of virus budding.

Amino Acid Motifs↗

PubMed2002

Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication.

Virions of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses contain conical cores consisting of a protein shell composed of the viral capsid protein (CA) surrounding an internal viral ribonucleoprotein complex. Although genetic studies have implicated CA in both early and late stages of the virus replication cycle, the mechanism of core disassembly following penetration of target cells remains undefined. Using quantitative assays for analyzing HIV-1 core stability in vitro, we identified point mutations in CA that either reduce or increase the stability of the HIV-1 core without impairing conical core formation in virions. Alterations in core stability resulted in severely attenuated HIV-1 replication and impaired reverse transcription in target cells with only minimal effects on viral DNA synthesis in permeabilized virions in vitro. We conclude that formation of a viral core of optimal stability is a prerequisite for efficient HIV-1 infection and suggest that disassembly of the HIV-1 core is a regulated step in infection that may be an attractive target for pharmacologic intervention.

Capsid↗