Biology subjects

Sloan, R. D.

Publications and source records attributed to Sloan, R. D..

3 recordsLinked to original sources

bioRxiv2026

The HIV-1 restriction factor RPRD2 does not inhibit transcription of HIV-1 or endogenous retroelements

A vital question in transcription regulation is how and to what extent transcription of different DNA species, including viral DNA, episomal DNA and endogenous retroelements, is differentially regulated to transcription of host genes. RPRD2 has previously been characterized as an HIV restriction factor that blocks reverse transcription. However, RPRD2 has also been characterized as a regulator of global transcription of host genes. Therefore, we hypothesized that RPRD2 may also regulate nascent transcription from HIV provirus and endogenous retroelements. First, we used a combination of experimental and computational methods to characterize the binding of RPRD2 to RNA and DNA:RNA hybrids. Using immunoprecipitation, we identified that RPRD2 interacts with both the transcription regulator PAF1 and the HUSH complex member TASOR, independently. Immunofluorescence revealed that GFP-RPRD2 localizes to foci in the nucleus, and these foci overlap with nuclear speckles. To measure the effect of RPRD2 on transcription, we used plasmid-borne HIV LTR-driven reporter constructs and observed that RPRD2 depletion increased transcription of constructs both with and without an intron. We next investigated transcription from integrated proviruses and found no effect of RPRD2 depletion using several different systems. Lastly, we measured transcription of endogenous retroelements and found that RPRD2 depletion did not affect transcription of LINE-1 or HERV-K. Finally, we investigated whether RPRD2 regulates production of IFN in response to nucleic acid species or affects transcription of IFN-stimulated genes. We found that depletion of RPRD2 had no effect on IFN production or ISG expression. Together, our findings demonstrate how regulation of transcription is not universal for host genes, integrated provirus, unintegrated plasmid and endogenous retroviruses, and confirmed that although RPRD2 governs cellular transcription, it does not regulate transcription of HIV-1 provirus or of endogenous retroelements.

microbiology↗

bioRxiv2023

Alternative splicing expands the antiviral IFITM repertoire in Chinese horseshoe bats

The interferon response is shaped by the evolutionary arms race between hosts and the pathogens they carry. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication, they restrict virus entry and are key players of the interferon response. Yet, little is known about IFITMs in other mammals. Here, we identified an IFITM gene in Chinese horseshoe bat, a natural host of SARS-coronaviruses, that is alternatively spliced to produce two IFITM isoforms. These bat IFITMs have conserved structures in vitro and differential antiviral activities against influenza A virus and coronaviruses including SARS- and MERS-coronavirus. In parallel with human IFITM1-3, the bat IFITM isoforms localize to distinct cellular compartments. Further analysis of IFITM repertoires in 205 mammals reveals that alternative splicing is a ubiquitous strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings showcase an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity.

microbiology↗

bioRxiv2018

HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity.

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 intact vpr is 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.\n\nImportanceFor 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-1 in 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 cells protein degradation machinery.

microbiology↗