APOBEC4 Enhances the Replication of HIV-1

APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters

Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors

International audienceBackground: Feline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untrans-lated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3.Results: To identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains.Conclusions: Here we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was developed. Our results show that HIV Vif binds human A3s differently than FIV Vif feline A3s. The linker insertion is suggested to form a homeo-box domain, which is unique to A3s of cats and related species, and not found in human and mouse A3s. Together, these findings indicate a specific and different A3 evolution in cats and human

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11.

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention

A4 enhances expression of CCR5-tropic HIV-1.

<p>(a) Increasing amounts of A4-HA expression plasmid were co-transfected with HIV-1 NL-BaL and immunoblot analysis of co-transfected 293T cells were performed. Immunoblots were probed with anti-p24 (capsid), anti-Vif, anti-HA and anti-tubulin (tub) antibodies. α, anti. (b) Infectivity of RT-normalized viral supernatant of the transfected cells from (a) were used to infect TZM-bl luciferase reporter cells. cps, counts per second. Data are represented as the mean with SD. Statistically significant differences between no A4 and A4 groups were analyzed using the unpaired Student’s t-test with GraphPad Prism version 5 (GraphPad software, San Diego, CA, USA). Validity of the null hypothesis was verified with significance level at α value = 0.05. NS: not significant.</p

Active site mutation has no influence on A4 activity.

<p>(a) Protein expression of A4-HA, A4-HA.E95Q and A4-HA.C134A detected by anti-HA immuno blot analysis, showing equal amounts of A4-HA and A4-HA.E95Q, but lack of A4-HA.C134A expression in transfected cells. (b) HIV-1 reporter virus (NL-Luc R^-E^-) was co-transfected with increasing amounts of expression plasmid for A4-HA.E95Q. Virus encoded luciferase activity in the transfected cells was enhanced by A4-HA.E95Q in a dose-dependent manner.</p

A4 enhances the expression of HIV-1.

<p>(a) HIV-1 genome expression plasmid was co-transfected with increasing amounts of HA-A4 expression plasmid, as indicated. A4 increases the production of HIV-1 particles as measured by the RT activity in the supernatant of the transfected cells. (b) Immunoblot analysis of virions and transfected 293T cells (same cells as in (a)). Immunoblots of virions and cell lysates were probed with anti-p24 (capsid) antibody. Anti-tubulin (tub) antibody served as loading control. α, anti. (c) RT concentrations in the supernatant of cells co-transfected with HA-A4 and HIV-1 plasmids relative to supernatant of cells co-transfected with empty vector and HIV-1, as in (a), summary of four independent experiments, median indicated. Evaluation of RT activity data was performed by means of a multifactorial analysis of variance (ANOVA).</p

A4 does not deaminate single stranded DNA.

<p>(a) Deamination activity of A4 was tested on two different oligonucleotide substrates containing nucleotide sequences CCCA or CCCG. The A3G-His fusion protein was incubated with CCCA and CCCG containing substrates and served as positive control for deamination resulting in 40-bp DNA fragments. Oligonucleotide CCUA served as a marker to denote the deaminated product after Eco147I cleavage; ND: not deaminated; D: deaminated. (b) Deamination experiment using TTCA containing oligonucleotide and GST-purified A4 proteins, RNAse A treatment was included; ND: not deaminated; D: deaminated. (c) Immuno blot analysis of cell lysates and virus lysate of A3G-HA, A3F-HA, 3xHA-A4 and HA-A4-ΔKK expressing cells and HIV virus like particles (VLP), respectively. Anti-HA staining indicates the presence of HA-tagged A3 and A4 proteins, while anti-p24 antibody detects HIV-1 capsid proteins. (d) Deamination assay using transfected 293T cell lysate (from experiment shown in (c)). RNAse A treatment was included; ND: not deaminated; D: deaminated. (e) Immuno blot analysis of cell lysate and immunoprecipitate (IP) fraction of A3 and A4 proteins. (f) Deamination assay using the immunoprecipitated APOBEC proteins (from experiment shown in (e)). RNAse A treatment was included; ND: not deaminated; D: deaminated.</p

Presence of A4 does not affect HIV-1 infectivity.

<p>HIV-1 reporter virus NL-Luc R^-E^- (VSV-G) was produced in 293T cells in the presence of increasing amounts of A4 (no tag) and A4-HA (C-terminal HA-tag). A4 and A4-HA increase in a dose-dependent manner both (a) the virus-encoded luciferase activity and (b) the expression of intracellular viral capsid (p24) in the transfected virus producing cells as demonstrated by immunoblot analysis (same cell lysates used in (a) and (b)). Error bars indicate standard deviation. (c) Immunoblot analysis of intracellular viral p24 (capsid) expression. Similar as in (a) and (b), NL-Luc R^-E^-/VSV-G was co-transfected with increasing amounts of HA-A4 plasmid (N-terminal HA-tag), as indicated. Immunoblots of cells were probed with anti-p24 (capsid) antibody. A4-HA expression in transfected cells was detected by immunoblotting using anti-HA antibody. Anti-tubulin (tub) antibody served as loading control. α, anti. (d) Relative viral luciferase activity in cells co-transfected with A4-HA and HIV-1 plasmids, as in (a). Summary of 28 independent experiments, median indicated. A4-HA was transfected in increasing amounts. (e) Equal volumes of supernatants of cells co-transfected with NL-Luc R^-E^-/VSV-G and increasing amounts of A4-HA were used to infect HOS cells. Intracellular luciferase activities were determined in infected cells; summary of 16 experiments (a subset of the experiments shown in (d)), median is indicated. (f) A subset of samples (seven experiments) used in (e) was quantified for RT concentrations. RT normalized supernatants of cells co-transfected with NL-Luc R^-E^-/VSV-G and increasing amounts of A4-HA were used to infect HOS cells. Intracellular luciferase activities determined in infected cells, median is indicated. (d—f) Statistical evaluation of reporter luciferase activity data was performed by means of a multifactorial ANOVA.</p