No abstract

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (MAbs) is challenging. Virus capture assays (VCAs) involving immobilized MAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing MAbs, including 2G12, 4E10, 2F5, Z13e1, and D5, will capture virion particles completely devoid of Env. We modified the VCA such that MAbs and virions are incubated in solution, and unbound MAbs are removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by MAbs to virions and allowed determination of apparent affinity constants in solution. Three important qualitative observations were further revealed. First, neutralizing MAbs 2F5, 4E10, and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype and that Env from HIV-1 JR-FL is more homogeneously trimeric than that from HIV-1 JR-CSF . Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular "bridge" between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.Eliciting neutralizing antibody (Ab) against human immunodeficiency virus type 1 (HIV-1) is a crucial but exceedingly difficult challenge in HIV-1 vaccine design (10, 32). The HIV-1 envelope glycoprotein (Env) is the specific target of all HIV-1 neutralizing Abs that have been identified to date (87,98). Env is produced as a gp160 precursor molecule that is cleaved by cellular proteases into a surface subunit, gp120, and a transmembrane subunit, gp41, which in the functional state of Env are assembled as noncovalent trimers of gp120-gp41 heterodimers (45, 91). The Env trimer engages host cell CD4 and coreceptor (CCR5 or CXCR4) through interaction with gp120, and this elicits conformational changes in gp41 that facilitate the subsequent fusion of virus and host cell membranes (29). However, native Env trimers coexist with distinct, nonfunctional forms of Env (34, 51, 65). These nonfunctional forms, including nontrimeric and aberrant disulfide-linked forms of Env, gp41 stumps from which gp120 has been shed, and uncleaved gp160, appear to be highly immunogenic but tend to elicit non-neutralizing antibodies (51,60,94).During the acute phase of natural infection, non-neutralizing Abs are commonly elicited, particularly to gp41 (83). Neutralizing Ab responses develop over time, but these tend to be is...

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.