These data suggested sufficient expression of Env gp140 NFL2, but that this percentage of well-ordered trimers in these preparations was relatively limited. to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses. Introduction The HIV-1 surface unit envelope glycoprotein (Env) trimer is usually comprised of the heterodimeric gp120 and gp41 subunits, which are the cleavage products of the trimeric Env precursor, gp160. The gp160 is usually cleaved by the endopeptidase furin in the Golgi complex of Env-expressing host cells (Checkley et al., 2011). Precursor cleavage GS-9620 is necessary for quaternary folding of the noncovalently associated gp120-gp41 subunits, affects the native conformation of the spike, restricts access of non-neutralizing antibodies (non-NAbs) and is required for viral entry into host cells (Herrera et al., 2005; McCune et al., 1988; Pancera and Wyatt, 2005; Ringe et al., 2013). Numerous studies demonstrate approximately 10-20% HIV-1-infected individuals generate broadly neutralizing antibodies (bNAbs) directed to Env (Stamatatos et al., 2009). The recent isolation of numerous bNAbs from HIV-1 infected individuals shows that, under the right circumstances, the human immune system can generate broadly effective antibodies. The biochemical and structural studies of these bNAbs yields insights about their cognate neutralizing determinants on Env and provides rationale for epitope-specific vaccine targeting. The bNAbs are directed against several accessible sites on gp120 and gp41, including the CD4 binding site (CD4bs), V1/V2 region glycans, glycans at the base of V3, and the gp41 membrane-proximal region (MPER) (West et al., 2014). Recently, other Env sites of vulnerability recognized by bNAbs which span both gp120 and gp41 subunits and involve N-linked glycans are described (Blattner et al., 2014; Falkowska et al., 2014; Huang et al., 2014; Scharf et al., 2014). Many of these bNAbs preferentially recognize Env trimers and are very GS-9620 useful probes for trimer design approaches, such as the one described here. To generate soluble, stable Env trimers designed to elicit neutralizing antibodies, gp140 oligomers were designed (Forsell et al., 2009). Generally, the gp140s are soluble versions of Env in which the cytoplasmic tail and transmembrane regions of gp41 are deleted, made up of full-length gp120 and most of the gp41 ectodomain. The natural association between gp120 and gp41 in the functional Env spike is usually non-covalent and labile, thereby GS-9620 recombinant expression of cleaved gp140s results in dissociation of the hetero-dimeric subunits. To address this problem, one approach alters the furin cleavage site to covalently link gp120 and gp41, preventing dissociation. Frequently, a trimeric motif (e.g., foldon) is usually attached at the C-terminus of gp41 to generate uncleaved, stable and soluble gp140 trimers; however, these trimers do not form well-ordered Env spike mimetics (Ringe et al., 2013; Tran et al., 2014). A second strategy introduces a disulfide linkage between gp120 and gp41 by genetically engineering cysteines at the interface of these subunits. The resulting covalently linked gp140s retain the natural cleavage site and are termed SOS soluble Env molecules (Binley et al., 2000). An additional HR1 helix destabilizing mutation in the gp41 ectodomain (i.e., I559P, now called SOSIP) greatly improves trimer formation (Sanders et al., 2002) and led to GS-9620 the recent atomic-level structural GS-9620 definition of BG505 SOSIP.664 (Julien et al., 2013; Lyumkis et al., 2013; Pancera et al., 2014; Sanders et al., 2013), hereafter referred to as BG505 SOSIP. Studies of both cell-surface Env and the soluble SOSIP trimers demonstrate that cleavage is usually important for native conformation based upon antigenic profiling and EM structural analysis (Pancera and Wyatt, 2005; Ringe et al., 2013). In these cases, only cleaved trimers preferentially present neutralizing determinants over non-neutralizing and form Mapkap1 well-ordered trimers. In contrast, the uncleaved gp140 trimers (e.g. gp140-foldon) generally present selected broadly neutralizing determinants but also present a variety of non-neutralizing epitopes, decreasing their desirability for rational vaccine designs aimed at presenting only neutralizing determinants to the humoral immune system. In the present study, we report a different strategy to generate native-like soluble, but uncleaved or cleavage-independent, gp140 trimers. We hypothesized that this cleaved termini of gp120 and gp41 are.