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