Lysines close to the Rous sarcoma virus late domain critical for budding

The release of retroviruses from the plasma membrane requires host factors that are believed to be recruited to the site of budding by the late (L) domain of the virus-encoded Gag protein. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a ubiquitin (Ub) ligase, and budding of this virus is dependent on Ub. RSV is similar to other retroviruses in that it contains approximately 100 molecules of Ub, but it is unique in that none of these molecules has been found to be conjugated to Gag. If transient ubiquitination of RSV Gag is required for budding, then replacement of the target lysine(s) with arginine should prevent the addition of Ub and reduce budding. Based on known sites of ubiquitination in other viruses, the important lysines would likely reside near the L domain. In RSV, there are five lysines located just upstream of the L domain in a region of the matrix (MA) protein that is dispensable for membrane binding, and replacement of these with arginine (mutant 1-5KR) reduced budding 80 to 90%. The block to budding was found to be on the plasma membrane; however, the few virions that were released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions, either within this region of MA or within the downstream p10 sequence. Moreover, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data argue that the phenotype of mutant 1-5KR is not due to a conformational defect. Consistent with the idea that efficient budding requires a specific role for lysines, human T-cell leukemia virus type 1, which does not bud well compared to RSV and lacks lysines close to its L domain, was found to be released at a higher level upon introduction of lysines near its L domain. This report strongly supports the hypothesis that ubiquitination of the RSV Gag protein (and perhaps those of other retroviruses) is needed for efficient budding.     

Membrane association induces a conformational change in the Ebola virus matrix protein

The matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for flexibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution. In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.     

The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly

In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant DeltaG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced approximately 10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.     

In vivo interference of Rous sarcoma virus budding by cis expression of a WW domain

For all enveloped viruses, the actual mechanism by which nascent virus particles separate or "pinch off" from the cell surface is largely unknown. In the case of retroviruses, the Gag protein drives the budding process, and the virus release step is directed by the late (L) assembly domain within Gag. A PPPPY motif within the L domain of Rous sarcoma virus (RSV) was previously characterized as being critical for the release of virions and shown to interact in vitro with the WW domain of Yes-associated protein (Yap). To determine whether WW domain-L domain interactions can occur in vivo, we attempted to interfere with the host cell machinery normally recruited to the site of budding by inserting this WW domain in different locations within Gag. At a C-terminal location, the WW(Yap) domain had no effect on budding, suggesting that the intervening I domains (which provide the major region of Gag-Gag interaction) prevent its access to the L domain. When positioned on the other side of the I domains closer to the L domain, the WW(Yap) domain resulted in a dramatic interference of particle release, and confocal microscopy revealed a block to budding on the plasma membrane. Budding was restored by attachment of the heterologous L domain of human immunodeficiency virus type 1 Gag, which does not bind WW(Yap). These findings suggest that cis expression of WW domains can interfere with RSV particle release in vivo via specific, high-affinity interactions at the site of assembly on the plasma membrane, thus preventing host factor accessibility to the L domain and subsequent virus-cell separation. In addition, they suggest that L domain-specific host factors function after Gag proteins begin to interact.     

A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.     

Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus.

The cytoplasmic domains of viral glycoproteins are often involved in specific interactions with internal viral components. These interactions can concentrate glycoproteins at virus budding sites and drive efficient virus budding, or can determine virion morphology. To investigate the role of the vesicular stomatitis virus (VSV) glycoprotein (G) cytoplasmic and transmembrane domains in budding, we recovered recombinant VSVs expressing chimeric G proteins with the transmembrane and cytoplasmic domains derived from the human CD4 protein. These unrelated foreign sequences were capable of supporting efficient VSV budding. Further analysis of G protein cytoplasmic domain deletion mutants showed that a cytoplasmic domain of only 1 amino acid did not drive efficient budding, whereas 9 amino acids did. Additional studies in agreement with the CD4-chimera experiments indicated the requirement for a short cytoplasmic domain on VSV G without the requirement for a specific sequence in that domain. We propose a model for VSV budding in which a relatively non-specific interaction of a cytoplasmic domain with a pocket or groove in the viral nucleocapsid or matrix proteins generates a glycoprotein array that promotes viral budding.     

Activation of the retroviral budding factor ALIX

The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.     

Domains and Rafts in Membranes – Hidden Dimensions of Selforganization

Both biomembranes and biomimetic membranes such as lipid bilayers withseveral components contain intramembrane domains and rafts.Macromolecules, which are anchored to the membrane but have no tendeney tocluster, induce curved nanodomains. Clustering of membrane componentsleads to larger domains which can grow up to a certain maximal size andthen undergo a budding process. The maximal domain size depends on theinterplay of spontaneous curvature, bending rigidity, and line tension.It is argued that this interplay governs the formation of bothclathrin-coated buds and caveolae. Finally, membrane adhesion often leadsto domain formation within the contact zone.     

流感病毒在脂筏上组装与出芽

流感病毒造成的季节性流行性疾病给全世界带来沉重的健康负担.近年来,甲型流感病毒的变种H5N1、H7N9给各国带来了很大危害.流感病毒属于正黏附病毒科,它的遗传物质由多个节段的负链RNA组成,其组装和出芽剪切生殖是一个涉及到多种病毒因子,多步骤、复杂的生化过程.流感病毒会使用宿主的细胞膜上的"脂筏"区域作为病毒出芽位点.首先病毒的两种糖蛋白NA蛋白、HA蛋白会在脂筏区域聚集,造成脂筏区膜变形弯曲,并且发动出芽的过程.接着,流感病毒基质蛋白M1的C端与HA、NA结合,其自身在脂筏区域开始多聚化并使膜向外弯曲形成原始病毒体的内部结构,接着招募病毒的核糖核蛋白复合物(VRNP)与M2蛋白,使组装的过程进一步完成.最后,M2蛋白会富集在原始病毒体的底部,完成膜的剪切和病毒体的释放.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.     

Role of ESCRT-I in retroviral budding

Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.     

Molecular Basis of the Potent Membrane-remodeling Activity of the Epsin 1 N-terminal Homology Domain

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal α-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.     

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230_GRIP fusion protein, we show binding specifically to a subset of membranes of the trans -Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230_GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230_GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230_GRIP binds to a specific population of vesicles distinct from those labelled for β-COP or γ-adaptin. The GFP-p230_GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans -Golgi network tubulo-vesicular carriers.     

Budding of retroviruses utilizing divergent L domains requires nucleocapsid

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.     

Growth dynamics of domains in ternary fluid vesicles

We have studied the growth dynamics of domains on ternary fluid vesicles composed of saturated (dipalmitoylphosphatidylcholine), unsaturated (dioleoylphosphatidylcholine) phosphatidylcholine lipids, and cholesterol using a fluorescence microscopy. The domain coarsening processes are classified into two types: normal coarsening and trapped coarsening. For the normal coarsening, the domains having flat circular shape grow in a diffusion-and-coalescence manner and phenomenologically the mean size grows as a power law of approximately t(2/3). The observed growth law is not described by a two-dimensional diffusion-and-coalescence growth mechanism following the Saffman and Delbrück theory, which may originate from the two-body hydrodynamic interactions between domains. For trapped coarsening, on the other hand, the domain coarsening is suppressed at a certain domain size because the repulsive interdomain interactions obstruct the coalescence of domains. The two-color imaging of the trapped domains reveals that the repulsive interactions are induced by the budding of domains. The model free energy consisting of the bending energy of domains, the bending energy of matrix, the line energy of domain boundary, and the translation energy of domains can describe the observed trapped coarsening. The trapping of domains is caused by the coupling between the phase separation and the membrane elasticity under the incompressibility constraint.     

Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding

The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.     

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.     

1H, 13C, and 15N resonance assignment of the N-terminal domain of Mason-Pfizer monkey virus capsid protein, CA 1-140

Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignment of the M-PMV NTD CA.     

Crystal structure of vesicular stomatitis virus matrix protein

The vesicular stomatitis virus (VSV) matrix protein (M) interacts with cellular membranes, self-associates and plays a major role in virus assembly and budding. We present the crystallographic structure, determined at 1.96 A resolution, of a soluble thermolysin resistant core of VSV M. The fold is a new fold shared by the other vesiculovirus matrix proteins. The structure accounts for the loss of stability of M temperature-sensitive mutants deficient in budding, and reveals a flexible loop protruding from the globular core that is important for self-assembly. Membrane floatation shows that, together with the M lysine-rich N-terminal peptide, a second domain of the protein is involved in membrane binding. Indeed, the structure reveals a hydrophobic surface located close to the hydrophobic loop and surrounded by conserved basic residues that may constitute this domain. Lastly, comparison of the negative-stranded virus matrix proteins with retrovirus Gag proteins suggests that the flexible link between their major membrane binding domain and the rest of the structure is a common feature shared by these proteins involved in budding and virus assembly.