Ultrasensitive quantification of HIV-1 cell-to-cell transmission in primary human CD4+ T cells measures viral sensitivity to broadly neutralizing antibodies

Abstract

HIV-1 efficiently replicates in vivo by direct transmission from infected to uninfected CD4+ T cells at confined local sites designated virological synapses (VSs). VSs are formed by cell junctions between HIV-1 envelope glycoproteins (Envs) on an infected cell and CD4 on an uninfected cell. These sites facilitate highly efficient viral transmission and contribute to HIV-1 evasion from neutralizing antibodies, but accurate quantification of the efficiency of cell-cell transmission is still challenging. Here, we developed an ultrasensitive HIV-1 cell- to-cell transmission assay that triggers the expression of the nanoluciferase (nluc) gene in target cells upon transmission and after reverse transcription of the HIV-1 RNA genome. The assay is based on insertion of the nluc gene in an antisense orientation into HIV-1 provirus; Nluc expression is blocked in virus-producing cells because the nluc gene contains an intron that can be efficiently spliced out only when mRNA is transcribed from the opposite (sense) strand. Thus, only sense mRNA that is spliced and subsequently reversed transcribed during transmission to target cells will support Nluc expression. Assay optimization resulted in a very low background, >99% splicing efficiency, high sensitivity and wide dynamic range for detection of cell-cell transmission in T cell lines and primary CD4+ T cells. The new reporter vector can detect cell-cell transmission using single-round viral vectors and HIV-1 molecular clones, which provide viral proteins of different HIV-1 strains, and reproducibly measures sensitivity of HIV-1 transmission to antibody neutralization in vitro. This assay will contribute to understanding fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study other retroviruses.