Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses

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.

RNA-Associated Early-Stage Antiviral Factor Is a Major Component of Lv2 Restriction

“…Also, it has been shown that disassembly occurs within an hour of fusion and is facilitated by reverse transcription (60)(61)(62). We have observed that cellular REAF is reduced within 1 h of a viral challenge, but importantly, levels are rapidly replenished an hour later (25). REAF associates with viral nucleic acid and restricts replication during reverse transcription (25).…”

“…We have observed that cellular REAF is reduced within 1 h of a viral challenge, but importantly, levels are rapidly replenished an hour later (25). REAF associates with viral nucleic acid and restricts replication during reverse transcription (25). We therefore suggested that the temporary reduction of the REAF protein level allows viruses to reverse transcribe in the absence of REAF-associated activity.…”

“…It is thought that optimal disassembly of the conical core is required for successful infection, as mutations interfering with core stability often result in a disturbance of reverse transcription kinetics (46,(49)(50)(51). We previously reported that REAF was transiently downmodulated shortly after infection (25). We hypothesized that unstable CAs will prematurely disassemble and expose reverse transcripts to REAF.…”

“…Further disabling reverse transcription, SAMHD1 depletes the deoxynucleoside triphosphate substrates required (24). RNA-associated early-stage antiviral factor (REAF) was reported to inhibit HIV and SIV replication during reverse transcription (25). REAF is intrinsically expressed and provides an initial line of defense against HIV and SIV infection.…”

Cellular minichromosome maintenance complex component 5 (MCM5) is incorporated into HIV-1 virions and modulates viral replication in the newly infected cells

“…These particles are derived from the viral capsid cores entering the target cell. Certain host cell proteins hijacked by virions from the virus-producing cells or recruited from the newly infected cell have been shown to stimulate [cyclophilin A (Briones et al, 2010; Luban, 2007; Luban et al, 1993; Schaller et al, 2011), heat shock protein 70 (Gurer et al, 2002), clathrin (Popov et al, 2011; Zhang et al, 2011), RNA helicase A (Jeang and Yedavalli, 2006; Roy et al, 2006), INI1/hSNF5 and Sin3a-HDAC1 complex (Sorin et al, 2009, 2006; Yung et al, 2004), importins α1 (Gallay et al, 1997) and α3 (Ao et al, 2010), importin 7 (Fassati et al, 2003; Zaitseva et al, 2009), TNPO3/transportin-SR2 (Christ et al, 2008; Krishnan et al, 2010), lens epithelium-derived growth factor (LEDGF) p75 (Ciuffi and Bushman, 2006; Llano et al, 2006; Maertens et al, 2003), barrier-to-autointegration factor (BAF) (Lin and Engelman, 2003), protein kinase A (PKA) (Giroud et al, 2013), eukaryotic elongation factor 1 (eEF1) (Warren et al, 2012)] or inhibit [apolipoprotein B mRNA editing enzyme catalytic (APOBEC) 3G (Bishop et al, 2008; Holmes et al, 2007b), APOBEC3F (Holmes et al, 2007a), Sterile alpha motif domain- and HD domain-containing protein (SAMHD) 1 (Hrecka et al, 2011; Laguette et al, 2011), Moloney leukemia virus (MOV) 10 (Furtak et al, 2010), RNA-associated early-stage anti-viral factor (REAF) (Marno et al, 2014)] the early events of HIV replication. Our published results of the proteomic analysis of HIV-1 cores (Santos et al, 2012) showed incorporation of multiple RNA- and DNA-binding proteins into the cores of virions assembled in CD4 + T cell (Sup-T1) and macrophage (PMA and vitamin D 3 activated THP-1) lines.…”