Mimicry of the NHR-helical trimers in class I viral fusion proteins for the discovery of antiviral therapeutics has been hampered because of the strong aggregation properties of synthetic NHR-based peptides taken out of their parent protein structure. To address this issue, researchers have long sought chemical interventions to reinforce the bioactive tertiary structure of NHR-based peptides and thereby restoring binding affinity towards their protein targets. One of the most established methods involves introducing an exogenous trimeric motif into the NHR-derived peptides to facilitate their folding into stable and soluble trimers. However, these modifications suffer from extra-large auxiliary protein domains, which may attenuate the binding of the NHR-trimer to their native ligand, and if used as immunogens or targets in mirror image phage display, the exogenous epitopes might induce undesired immune responses and divert the real conformation of the NHR trimer to the prefusogenic state. Inspired by the tractability of coiled coils and the ultrastability provided by isopeptide bond crosslinks present in a range of bacterial surface proteins, we herein report an effective strategy to recapitulate NHR trimeric coiled coils in HIV-1 membrane fusion protein, containing no any exogenous trimerization motif. One of these NHR-trimers, (N36M)3, exhibited highly potent inhibitory activity against HIV-1 infection, exceptional resistance to proteolysis, and effective native ligand-binding capability. This study proves for the first time that this stabilization strategy can be used to yield a trimeric coiled coil-based polypeptide or protein without containing an exogenous trimerization motif as highly potent NHR-peptide-based fusion inhibitors against HIV-1 and other class I enveloped viruses.