Background
It has been demonstrated that over 95% of provirus present in the peripheral blood of people living with HIV on combination antiretroviral therapy are defective. Defective viruses have long been considered biologically irrelevant, as they cannot encode infectious viruses. However, recent studies have shown that these defective viruses are translationally active, raising potential concerns for current HIV therapies.Therefore, there is a need for a better understanding of the biological function of defective HIV proviruses.
Methods
Previously subcloned cell lines derived from H9MN were used in this study. The H9-FI harbored an intact full-length HIV-1 provirus, the H9-FD contained a full-length defective provirus with a 1-bp inserted frameshift, and the H9-SD possessed a 2.4-kb internal deletion. Cytopathic effect was determined using H9-FD or H9-SD cells co-culturing with MT-2 cells. Extracellular vehicles (EVs) were isolated from cell culture supernatants of H9-FD or H9-SD using ultracentrifugation. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis were used to characterize the EVs. A CodePlex secretome proteomics workflow was used to measure cytokine inductions.
Results
SEM analysis confirmed 25% of viral particles budding from H9-FD cells, with 75% of these particles being fully released, a level similar to that observed in H9-FI. Additionly, TEM images confirmed that more than 90% of EVs from H9-FD were virus-like particles (VLPs), with the remainder being typical EVs. Of the VLPs, approximately 5 to 10% were mature, while 90 to 95% were immature particles. In contrast, no VLPs were identified in the EVs from H9-SD. Of interest, cell killing of MT-2 cells was observed only when co-cultured with H9-FD, not with H9-SD cells, indicating that VLPs are critical in causing the cytopathic effect. Stimulating human primary T cells with a mixture of EVs, but not with individual EVs, was found to induce the production of multiple cytokines, including IFN-γ, IL-10, IL-15, IL-17A, IL-2, IL-4, IL-5, IP-10, sCD137, and TNF-α.
Conclusions
The present study demonstrated that a mixture of EVs produced by cells harboring defective HIV provirus is a potent stimulator of human primary T cells and that even EVs without VLPs can still contribute to initiating inflammatory cascades in the human body. This finding provides new insight: HIV-1 defective proviruses are not silent and may play a critical role in stimulating innate immune responses.