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The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intactvpris more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.ImportanceFor at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication in human cells is restricted at early postentry steps by host inhibitory factors. We previously described and characterized an early-phase restriction of HIV-1 and -2 replication in human cell lines, primary macrophages, and peripheral blood mononuclear cells. The restriction was termed lentiviral restriction 2 (Lv2). The viral determinants of Lv2 susceptibility mapped to the HIV-2 envelope (Env) and capsid (CA). We subsequently reported a whole-genome small interfering RNA screening for factors involved in HIV that identified RNA-associated early-stage antiviral factor (REAF). Using HIV-2 chimeras of susceptible and nonsusceptible viruses, we show here that REAF is a major component of the previously described Lv2 restriction. Further studies of the viral CA demonstrate that the CA mutation I73V (previously called I207V), a potent determinant for HIV-2, is a weak determinant of susceptibility for HIV-1. More potent CA determinants for HIV-1 REAF restriction were identified at P38A, N74D, G89V, and G94D. These results firmly establish that in HIV-1, CA is a strong determinant of susceptibility to Lv2/REAF. Similar to HIV-2, HIV-1 Env can rescue sensitive CAs from restriction. We conclude that REAF is a major component of the previously described Lv2 restriction. IMPORTANCE Measures taken by the host cell to combat infection drive the evolution of pathogens to counteract or sidestep them. The study of such virus-host conflicts can point to possible weaknesses in the arsenal of viruses and may lead to the rational design of antiviral agents. Here we describe our discovery that the host restriction factor REAF fulfills the same criteria previously used to describe lentiviral restriction (Lv2). We show that, like the HIV-2 CA, the CA of HIV-1 is a strong determinant of Lv2/REAF susceptibility. We illustrate how HIV counteracts Lv2/REAF by using an envelope with alternative routes of entry into cells.

The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years.

Background 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. Results We 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. Conclusions This 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.

Background The Polymerase II-Associated Factor 1 (PAF1) is a cellular protein with essential roles in transcription activation and repression, polyA tail cleavage, regulation of RNA polymerase elongation, regulation of gene expression, cell cycle control and mRNA quality surveillance[1]. It is part of a multicomponent complex together with other cellular proteins including SKI8, LEO1, CDC73, RTF1 and CTR9 [1]. We described that PAF1 has antiviral effect against HIV-1 that manifests early in the replication cycle [2]. Marazzi et al. described that PAF1 also inhibits influenza virus infection suggesting that PAF1 could have a broad antiviral activity [3]. Localisation of endogenous PAF1 is mainly in the nucleus but also in the cytoplasm. We propose three hypotheses: that PAF1 acts from the cytoplasm or the nucleus or both.