The Heptad Repeat 2 Domain Is a Major Determinant for Enhanced Human Immunodeficiency Virus Type 1 (HIV-1) Fusion and Pathogenicity of a Highly Pathogenic HIV-1 Env

Abstract

Human immunodeficiency virus type 1 (HIV-1)-mediated depletion of CD4؉ lymphocytes in an infected individual is the hallmark of progression to AIDS. However, the mechanism for this depletion remains unclear. To identify mechanisms of HIV-1-mediated CD4 T-cell death, two similar viral isolates obtained from a rapid progressor patient with significantly different pathogenic phenotypes were studied. One isolate (R3A) demonstrates enhanced pathogenesis in both in vivo models and relevant ex vivo lymphoid organ model systems compared to another isolate, R3B. The pathogenic determinants were previously mapped to the V5-gp41 envelope region, correlating functionally with enhanced fusion activity and elevated CXCR4 binding affinity. To further elucidate specific differences between R3A and R3B within the V5-gp41 domains that enhance CD4 depletion, R3A-R3B chimeras to study the V5-gp41 region were developed. Our data demonstrate that six residues in the ectodomain of R3A provide the major determinant for both enhanced Env-cell fusion and pathogenicity. Furthermore, three amino acid differences in the heptad repeat 2 (HR-2) domain of R3A determined its fusion activity and significantly elevated its pathogenic activity. The chimeric viruses with enhanced fusion activity, but not elevated CXCR4 affinity, correlated with high pathogenicity in the thymus organ. We conclude that the functional domain of a highly pathogenic HIV-1 Env is determined by mutations in the HR-2 region that contribute to enhanced fusion and CD4 T-cell depletion.

HIV-1 Env and Nef Cooperatively Contribute to Plasmacytoid Dendritic Cell Activation via CD4-Dependent Mechanisms

“…The R3A genome was digested with EcoRI to obtain two principal fragments: 5= and 3=. The 5= fragment containing the 5= long terminal repeat (LTR), gag, pol, vif, and the 5= part of vpr was subcloned into the p83-2 plasmid (NIH AIDS Research and Reference Reagent Program) (30). The 3= fragment containing the 3= part of vpr, tat, rev, vpu, env, nef, and the 3= LTR was subcloned into the p83 plasmid (with EcoRI and XhoI) (NIH AIDS Research and Reference Reagent Program) (30).…”

“…Isolation of Env from primary isolates and cloning of NL4-R3A (R3A) and NL4-R3B (R3B) proviral plasmids, as well as their Nefmutated versions, have been previously described (28,30). Virus stocks were generated by transfection of the proviral plasmids into HEK293T cells by the standard calcium chloride method.…”

“…Enhanced CD4 binding, entry, and in vitro cytopathicity of R3A were mapped to V1V2 of the Env protein. Enhanced CXCR4 binding affinity and fusion activity were mapped to the V5-gp41 domains (29,30). Subsequently, R3A, but not R3B, was able to induce high levels of IFN-I in human thymus, which contributed to the greater depletion of human T cells (31).…”

Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket

“…It is generally accepted that substitutions in the inhibitor-binding site of NHR helix are primary mutations that dominate resistance, whereas substitutions in the CHR helix serve as secondary mutations that may compensate the delayed fusion kinetics of virus caused by primary resistance substitutions (21,38,45,46,48). The N126K mutation, located near the pocket-binding domain (PBD) of CHR helix, has been frequently observed during escape selection against diverse fusion inhibitors (8,14,(19)(20)(21)49) and in prolonged T20-containing clinical therapy (9-12, 45, 50, 51).…”

“…While the amino acid substitutions on the inhibitor-binding site of gp41 are considered primary mutations responsible for resistance, the CHR mutations have been recognized as secondary or compensatory mutations that can improve the kinetics of viral fusion (21,45,46). Previous studies demonstrate that N126K not only enhances the interaction between the NHR and CHR helices but also mediates a mild resistance to diverse fusion inhibitor peptides (20,21,38).…”