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Recent studies implicate the vacuolar protein-sorting pathway in the transport of the retroviral structural precursor (Gag) protein to its budding site on the plasma membrane of infected cells. This exploitation of the cellular endocytic trafficking machinery to release viral particles could lead to the identification of virus-specific modulators and provide opportunities to design new targeted anti-viral agents. [ABSTRACT FROM AUTHOR]

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cellexpressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

A retroviral capsid (CA) protein consists of two helical domains, CAN and CAC, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr20 to Pro17. This coil to helix transition involves the conserved sequence of Thr16-Pro17-Arg18 (Ser16-Pro17-Arg18 in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CAN oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg18, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CAN oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CAN domain oligomerization, independent to the faster EIAV-CACdomain dimerization. [ABSTRACT FROM AUTHOR]

Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Δp2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Δp2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Δp2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin- tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements. [ABSTRACT FROM AUTHOR]

Discusses the evolution of the functional diversity in cupin proteins. Characteristics of cupins; Details on the active site of cupins; Phylogeny of cupins.

The Gag protein of avian sarcoma virus (ASV) lacks anN-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MAdomain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipaseC (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to thePMand budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to thePMwhen substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding. [ABSTRACT FROM AUTHOR]

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2. We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMRbased liposome-binding assay, we show that binding ofMA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding ofMA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly. [ABSTRACT FROM AUTHOR]

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub 1), here we show that it can also bind several diubiquitins (K48-Ub 2 , N-Ub 2 , and K63-Ub 2), with a preference for K63-linked Ub 2. The NMR structure of the Tsg101:K63-Ub 2 complex showed that while the Ub 1 -binding site accommodates the distal domain of Ub 2 , the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo. [Display omitted] • The Tsg101:K63-linked di-ubiquitin structure is solved by NMR • Pseudocontact shifts are used to dock and refine the structure of the complex • Lanthanide incorporation into specific ubiquitin (Ub) could distinguish between two Ub domains • Two different conformations of the diubiquitin are required to satisfy the NMR data Strickland et al. determine the structure of the K63 linked diubiquitin bound to Tsg101 UEV domain. The structure reveals the distal ubiquitin binds the monoubiquitin site on UEV, whereas the proximal ubiquitin can occupy two distinct sites. The two conformations of the diubiquitin on UEV have different functional consequences. [ABSTRACT FROM AUTHOR]

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal β-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the β-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus 13-hairpin by disrupting the salt bridge between Pro¹ and Asp54 that stabilizes the β-hairpin. NMR 15N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the β-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the β-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation. [ABSTRACT FROM AUTHOR]