Abstract
BackgroundThe discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. We identified RNA-associated Early-stage Anti-viral Factor (REAF) using a whole genome siRNA screen for restriction factors to human immunodeficiency virus (HIV) that act in the early phase of viral replication.ResultsWe observed more than 50 fold rescue of HIV-1 infection, using a focus forming unit (FFU) assay, following knockdown of REAF by specific siRNA. Quantitative PCR was used to show that REAF knockdown results in an increase of early and late reverse transcripts which impacts the level of integration. REAF thus appears to act at an early stage of the viral life cycle during reverse transcription. Conversely when REAF is over-expressed in target cells less infected cells are detectable and fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF associates with viral nucleic acids and may act to prevent reverse transcription.ConclusionsThis report firmly places REAF alongside APOBECs and SAMHD1 as a potent inhibitor of HIV replication acting early in the replication cycle, just after cell entry. We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen.
Highlights
The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity
HeLa-CD4 cells were transfected with an siRNA library targeting 19,121 human genes and challenged with an human immunodeficiency virus (HIV)-189.6R pseudovirus carrying a GFP reporter gene (HIV-1 gag/pol/tat and rev, HIV-2 MCR Env)
To quantify the ability of RNA-associated Early-stage Anti-viral Factor (REAF) to rescue infection, HeLa-CD4 cells were challenged with dual tropic wild type HIV-189.6 following siRNA knockdown
Summary
The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. We identified RNA-associated Early-stage Anti-viral Factor (REAF) using a whole genome siRNA screen for restriction factors to human immunodeficiency virus (HIV) that act in the early phase of viral replication. Once HIV enters the cytoplasm its genomic RNA is reverse transcribed by the virally encoded reverse transcriptase (RT), resulting in hybrid RNA:DNA intermediates. Once reverse transcription is complete the pre-integration complex (PIC) containing the double stranded (ds)DNA is formed and integrated into the genome of the host cell. This process is inhibited by the TRIM28 (KAP1)/SETDB1 complex [6,7]. A plasma membrane located restriction factor tetherin/BST-2/CD317, prevents viruses from leaving the cell at the late budding stage of the life cycle [9]
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