A virus-envelope paired competitive assay to study entry efficiency of human immunodeficiency virus type 1 in vitro

The efficiency of the human immunodeficiency virus type-1 (HIV-1) to enter cells is defined primarily by amino acid exchanges in the external glycoprotein gp120 and in, especially its highly variable V3 loop region. To study entry efficiency of HIV-1 a competitive viral entry assay was developed, to be comprised of infectious virus as well as soluble gp120 (sgp120) as an entry competitor. Entry of viruses using the coreceptor CXCR4 was reduced by adding CXCR4-tropic sgp120 (X4-sgp120) SF2 or LAV expressed in the baculovirus system or by adding X4-sgp120 from NL-952 and NL-V3A virus mutants produced in a HeLa-P4 cell culture expression system. Adding X4-sgp120 into a CCR5-specific infection assay revealed that X4-sgp120 enhanced the infection of CCR5-tropic virus. Furthermore, the role of the V3 loop N-glycan g15 on entry efficiency was studied using virus mutants and sgp120 with different N-glycosylation and different coreceptor usage. These experiments showed that viral entry of R5-tropic viruses lacking the N-glycan g15 within the V3 loop was inhibited by CCR5-tropic sgp120 harboring the g15 N-glycan. Altogether, the data demonstrate that HIV-1 entry efficiency can be studied easily by using sgp120 as an internal control or by using autologous or heterologous sgp120-virus pairs.