The budding of enveloped viruses from cellular membranes is believed to be dependent on the specific interaction between transmembrane spike proteins and cytoplasmic core components of the virus. We found that the cytoplasmic domain of the E2 transmembrane spike glycoprotein of Semliki Forest virus contains two essential determinants which are absolutely needed for budding. The first constitutes a single tyrosine residue in the context of a direct pentapeptide repeat. The tyrosine could only partially be substituted for other residues with aromatic or bulky hydrophobic side chains, although these immediately reverted to the original genotype. The second determinant involves palmitylated cysteine residues flanking the tyrosine repeat motif. The function of these is probably to anchor the tail against the inner surface of the membrane so that the tyrosine‐containing motif is properly presented to the nucleocapsid. This is the first example where a membrane virus employs a tyrosine signal for the selective incorporation of spike proteins into budding structures.

L.Xing and K.Tjarnlund contributed equally to this workReceptor binding to human poliovirus type 1 (PV1/M) and the major group of human rhinoviruses (HRV) was studied comparatively to uncover the evolution of receptor recognition in picornaviruses. Surface plasmon resonance showed receptor binding to PV1/M with faster association and dissociation rates than to HRV3 and HRV16, two serotypes that have similar binding kinetics. The faster rate for receptor association to PV1/M suggested a relatively more accessible binding site. Thermodynamics for receptor binding to the viruses and assays for receptor-mediated virus uncoating showed a more disruptive receptor interaction with PV1/M than with HRV3 or HRV16. Cryoelectron microscopy and image reconstruction of receptor-PV1/M complexes revealed receptor binding to the 'wall' of surface protrusions surrounding the 'canyon', a depressive surface in the capsid where the rhinovirus receptor binds. These data reveal more exposed receptor-binding sites in poliovirus than rhinoviruses, which are less protected from immune surveillance but more suited for receptor-mediated virus uncoating and entry at the cell surface.

Receptor priming of low-pH-triggered virus entry has been described for an enveloped virus (15). Here we show with major group human rhinoviruses (HRV) and its intercellular adhesion molecule-1 receptor that nonenveloped viruses follow this novel cell entry principle. In vitro the receptor primed HRV for efficient uncoating at mild low pH (5.5 to 6.0). Agents preventing endosomal acidification reduced or blocked rhinovirus cell infection, while nocodazole had no effect on infection of any serotype tested. The entry inhibitory effect of lysosomotropic agents was overcome by exposing cell-internalized HRV to mild low pH (5.5 to 6.0). We therefore conclude that receptor priming of major group HRV must occur in vivo as well. Cooperation of a cellular receptor and low pH in virus uncoating will polarize the exit of the genome to the receptor-bound, membrane-proximal region of the virus particle during acidification of endosomes. This process must be required for efficient penetration of the cellular membrane by viruses.Entry of viruses into host cells requires binding to one or several cell surface receptors and subsequent penetration of the cellular membrane. The penetration or entry process in enveloped viruses occurs by fusion of virus and cell membranes (21), while in nonenveloped viruses the process requires local disruption of the bilayer (3). For most viruses, cell entry is mediated by either receptor (pH independent) or low pH. Recently, however, it was shown that entry of the retrovirus avian leukosis virus (ALV) required both receptor and low pH, providing a novel principle for entry of enveloped viruses (15). Binding of ALV to its cellular receptor converts the viral envelope protein into a metastable form sensitive to low pH so that subsequent virus internalization and endosomal acidification triggers membrane fusion and release of the viral capsid into the cell cytoplasm.Cell entry and receptor recognition have been extensively studied in picornaviruses, a large family of nonenveloped viruses responsible of several human and animal diseases (19). Picornaviruses are constituted by an icosahedral protein capsid built by 60 protomers assembled in 12 pentamers, with a singlestranded RNA genome closely packed inside. Cell entry requires uncoating or exit of the RNA from the capsid, which presumably moves to the cell cytoplasm through a membrane pore generated by hydrophobic capsid polypeptides (3). Receptor-mediated uncoating of picornaviruses at neutral pH was first described for poliovirus (12). Poliovirus does not require a low-pH step or endocytosis for cell entry (8, 16), so multimeric binding of the virus to the receptor must trigger the molecular events leading to virus penetration (18). The poliovirus entry pathway differs from that described for human rhinovirus serotype 2 (HRV2), a member of the minor group of HRV, which bind to receptors belonging to the low-density lipoprotein (LDL) family (10). HRV2 requires both endocytosis and a low-pH step for efficient uncoating and cell entry (1, 2). The LDL...

The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N-and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.retrovirus spike | receptor triggering | cryo-EM | single particle imaging | EMAN T he HIV-1 spike facilitates entry of the virus into the cell by mediating fusion between the viral and the cell membranes. It also represents the target for neutralizing antibodies of the host. The spike is assembled from three copies of a transmembrane precursor glycoprotein, gp160, in the endoplasmic reticulum of the infected cell and is activated by a series of structural transitions (1-3). When the spike passes trans Golgi, on its way to the cell surface, gp160 is cleaved by furin into gp41 and gp120, which remain noncovalently linked (4). The cleavage positions the fusion peptide at the N terminus of gp41 and primes the spike for fusion activation. In the virus the gp120 subunits suppress the fusion activity of the gp41 subunits until structurally changed by receptor interactions, first with CD4 and then with the chemokine coreceptor (5-9). The gp41 subunits induce membrane fusion through refolding into a more stabile form. According to the prevailing model, the gp41 first targets the cell membrane with its fusion peptide and then folds back on itself dragging the virus and the cell membranes together for fusion (10). Characteristic for the gp41 ectodomain is two α-helical regions (N and C helices) separated by a small disulfide loop, CX 5 C. Peptides corresponding to the helical regions form a stable complex in solution and the crystal structure shows a bundle of six helices, where thr...

Alphaviruses are taken up into the endosome of the cell, where acidic conditions activate the spikes for membrane fusion. This involves dissociation of the three E2-E1 heterodimers of the spike and E1 interaction with the target membrane as a homotrimer. The biosynthesis of the heterodimer as a pH-resistant p62-E1 precursor appeared to solve the problem of premature activation in the late and acidic parts of the biosynthetic transport pathway in the cell. However, p62 cleavage into E2 and E3 by furin occurs before the spike has left the acidic compartments, accentuating the problem. In this work, we used a furin-resistant Semliki Forest virus (SFV) mutant, SFV SQL , to study the role of E3 in spike activation. The cleavage was reconstituted with proteinase K in vitro using free virus or spikes on SFV SQL -infected cells. We found that E3 association with the spikes was pH dependent, requiring acidic conditions, and that the bound E3 suppressed spike activation. This was shown in an in vitro spike activation assay monitoring E1 trimer formation with liposomes and a fusion-from-within assay with infected cells. Furthermore, the wild type, SFV wt , was found to bind significant amounts of E3, especially if produced in dense cultures, which lowered the pH of the culture medium. This E3 also suppressed spike activation. The results suggest that furin-cleaved E3 continues to protect the spike from premature activation in acidic compartments of the cell and that its release in the neutral extracellular space primes the spike for low-pH activation.The alphavirus spike is a trimer of the E1-E2 heterodimer (6,19,42,46). Both subunits are transmembrane glycoproteins (12). E1 carries the membrane fusion function of the virus, while E2 binds the virus to a still ill-defined receptor structure(s) on the cell surface (16,17,32). E2 also controls the fusion function of E1 so that it does not occur before the virus has entered the endosome (23, 38). There, the acidic pH dissociates the heterodimer and allows the E1 to interact with the endosomal membrane via its fusion loop (16,33). This results in E1 homotrimerization and subsequent jack knife like back folding, which brings the viral and the endosomal membranes together for fusion (16,34). The oligomerization of the spike subunits into the heterodimer and the subsequent trimerization of the heterodimers into spikes take place in the rough endoplasmic reticulum of the infected cell (20,25,45). The spikes are then transported to the cell surface via the Golgi complex and apparently also the early endosome (3, 28). At the cell surface, the spikes interact with the viral nucleocapsid and with each other, driving budding of virus particles with T ϭ 4 icosahedral symmetry (6, 10, 31). The fact that the heterodimer is made as an acid-resistant E1-p62 precursor appears to be an elegant solution to avoid premature activation when passing the acidic conditions of the Golgi complex and the early endosome (8, 9, 11, 33). However, the cleavage of the p62 subunit into E2 and the small perip...