Interferon (IFN) is a key modulator of the immune response against pathogens. One of its many functions is to induce the expression of several viral restriction factors. In particular, our project is focussed on Interferon-induced trans-membrane proteins (IFITM), whose primary role is to block early stages of viral replication.
IFITMs mediated restriction occurs during viral entry before viral membrane fusion takes place.
HIV-1 enters cells through engagement of its envelope glycoprotein with CD4 and a co-receptor, CCR5 (R5) or CXCR4. Importantly, R5 viruses are exclusively associated with primary HIV-1 infection and have shown to be resistant to the antiviral activity of IFITMs.
We are employing the 293-Affinofile system to investigate the relationship between IFITM activity in conjunction with the expression levels of CD4 and CCR5 on HIV-1 infectivity. 293-Affinofiles is a dual inducible cell line where CD4 and CCR5 expression can be induced independently and simultaneously.
Different IFITMs were overexpressed or shRNA knock-down in the Affinofile cell line. Neither overexpression nor the silencing of IFITMs affected the induction levels of CD4 and CCR5 expression. HIV-1 R5-tropic ADA and JR-CSF were resistant to IFITM 1 and 2 mediated restriction. Surprisingly, the silencing of IFITM1/2 differentially restricts ADA, JR-CSF and YU2 infection. However, infectivity levels were restored when increasing CD4 expression.
IFITM proteins are localised within different subcellular compartments; therefore, the pattern of IFITM restriction implies that ADA, JR-CSF and YU2 enter cells at spatially distinct localizations. To investigate this possibility, with use of an HIV-1 Fusion Assay and a membrane-impermeable viral fusion inhibitor, we will examine if the susceptibility to IFITM corresponds to a differential virus trafficking and localisation of viral fusion site.
Understanding how these restriction factors block viral spread has important implications for understanding HIV-1 cell tropism and may allow devising novel antiviral strategies.