In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

We recently described a system for heterologous gene expression in a variety of mammalian cell types that is based on an efficiently replicating Semliki Forest virus (SFV) variant in which an RNA encoding a foreign protein replaces the RNA that normally encodes the viruses' structural polyprotein. Although expression levels are sufficiently high for many purposes, in general they are only 10% of the level of the polyprotein in a wild type SFV infection. Here we show that the first 102 bases of the viral capsid gene function as a translational enhancer, and that SFV vectors incorporating this RNA increase heterologous protein synthesis to the level of wild type polyprotein.

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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...

Assembly of human immunodeficiency virus type 1 (HIV-1

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...

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.three-dimensional structure | retrovirus | spike protein T he spike protein Env on the surface of the retrovirus murine leukemia virus (MLV) matures by two proteolytic cleavage events mediated by cellular furin and the viral protease (1-3). Env is composed of an 80-kDa transmembrane precursor protein in the rough endoplasmic reticulum of the infected cell (4, 5). Here it trimerizes before it is routed to the cell surface for assembly with the internal Gag and GagPol precursors into virus particles in a budding process (6). The furin cleavage of Env occurs in the trans-Golgi, and it separates the surface (SU) subunit from the transmembrane (TM) (Pr15E) subunit. This cleavage also releases the viral fusion peptide at the N-terminal end of the TM. The viral protease cleavage occurs after virus assembly in the newly formed particle. It removes a 16-residuelong peptide, the R-peptide from the C terminus of the TM, forming p15E (7). Not until this second cleavage is completed is Env primed for receptor-mediated triggering into further conformations that can direct virus entry through fusion of the viral membrane with the cell membrane (8, 9). The viral protease also cleaves the Gag and GagPol precursors into their mature proteins and enzymes.The structure of the MLV Env has been studied by cryoelectron microscopy (cryo-EM) both in intact particles and as solubilized trimers (10, 11). It reveals a remarkably open structure with separated legs. The protomer of the solubilized Env is formed from three protrusions-upper, middle, and lower. Together, they encage a large central cavity, with the top pro...

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