Principal HIV-1 entry pathways and a functional strategy to determine the entry site. (A) HIV-1 binds to CD4 along with a coreceptor and either undergoes fusion with the plasma membrane (PM) or with an endosomal membrane (Endo). While a C-peptide only inhibits fusion of non-internalized particles that have not fused at the time of its addition, low temperature (referred to as a temperature block, TB) is able to inhibit both PM and Endo fusion. (B) The time course of HIV-1 escape from fusion inhibitors during entry into and fusion with target cells. The virus sensitivity to C-peptide and TB would change at the similar rate, if fusion occurs at the plasma membrane (PM). By contrast, viruses entering through the endosomal route would acquire resistance to C-peptide but remain sensitive to the TB until they undergo fusion with an endosomal membrane.