The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Abstract

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity. IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

The fusion peptide proximal region of the HIV-1 envelope glycoproteins regulates exposure of immunogenic epitopes at the trimer base

“…We expect that the changes in the FPPR induce similar release of structural constraints that maintain the trimer in a closed conformation. Based on proximity of the FPPR to the CHR, is it likely that the FPPR substitutions affect the interaction between the two domains (28). The nature of the association between the MPER and other components of gp41, which can explain the structural basis for our observations, remains to be clarified.…”

“…In the unliganded form of Env (i.e., not bound to CD4), the FPPR appears to be conformationally flexible (25,26). Nevertheless, substitutions at some FPPR positions increase detachment of gp120 from virions (27,28), supporting the notion that this region contributes to structural stability of the trimer. We analyzed the historical changes in amino acid sequence of the FPPR among clade B viruses and tested the emerging variants for their fitness and antigenicity.…”

Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors

“…Specifically, the N36/C34 complex showed 89% α-helices with a melting temperature ( T m ) value of 63 ℃, whereas the N36/C34 N145A complex showed 81% α-helices with a T m of 51 ℃. Because the recent studies suggested that the interactions between the FPPR of NHR and the TRM of CHR also critically determine the NHR-CHR interactions and HIV-1 entry [14,22,34], we further used the NHR-derived peptide N39, the inhibitor T20, and its N145A mutant as surrogates. As shown in Figure 4C-D, the N39/T20 complex displayed 53% α-helices with a T m of 43 ℃, but the α-helical content and T m of the N39/T-20 N145A complex could not be defined, which suggested that the N145A substitution disrupted the helical interaction between N39 and T20.…”

Reverted HIV-1 Mutants in CD4 + T-Cells Reveal Critical Residues in the Polar Region of Viral Envelope Glycoprotein

“…Previous studies suggested that the PR participates in Env peptide-based membrane fusion ( 5 – 9 ); however, the function of the PR is not fully understood. We have reported that the PR is highly conserved and determines viral fusion and infectivity ( 10 ). We characterized three PR mutants ( 10 ) containing S534P/T536A, S534P/T536A/T538A or S534P, named M1, M3 or M4, respectively ( Fig.…”

“…We have reported that the PR is highly conserved and determines viral fusion and infectivity ( 10 ). We characterized three PR mutants ( 10 ) containing S534P/T536A, S534P/T536A/T538A or S534P, named M1, M3 or M4, respectively ( Fig. 1A ).…”