Study of in vitro phosphorylation of histones H3, H4 and of the non-acetylated and acetylated tetramers (H3–H4)2

Granyer Giralt, JosepPrimer pla de l'obra. Coneguda com el Vedell o el Toro, en actitud de meditar. Mesura 2,42 x 0,62 x 0,76 metres i és de bronze

Impact of chemotherapy for HIV-1 related lymphoma on residual viremia and cellular HIV-1 DNA in patients on suppressive antiretroviral therapy

The first cure of HIV-1 infection was achieved through complex, multimodal therapy including myeloablative chemotherapy, total body irradiation, anti-Thymocyte globulin, and allogeneic stem cell transplantation with a CCR5 delta32 homozygous donor. The contributions of each component of this therapy to HIV-1 eradication are unclear. To assess the impact of cytotoxic chemotherapy alone on HIV-1 persistence, we longitudinally evaluated low-level plasma viremia and HIV-1 DNA in PBMC from patients in the ACTG A5001/ALLRT cohort on suppressive antiretroviral therapy (ART) who underwent chemotherapy for HIV-1 related lymphoma without interrupting ART. Plasma HIV-1 RNA, total HIV-1 DNA and 2-LTR circles (2-LTRs) in PBMC were measured using sensitive qPCR assays. In the 9 patients who received moderately intensive chemotherapy for HIV-1 related lymphoma with uninterrupted ART, low-level plasma HIV-1 RNA did not change significantly with chemotherapy: median HIV-1 RNA was 1 copy/mL (interquartile range: 1.0 to 20) pre-chemotherapy versus 4 copies/mL (interquartile range: 1.0 to 7.0) post-chemotherapy. HIV-1 DNA levels also did not change significantly, with median prechemotherapy HIV-1 DNA of 355 copies/106 CD4+ cells versus 228 copies/106 CD4+ cells post-chemotherapy. 2-LTRs were detectable in 2 of 9 patients pre-chemotherapy and in 3 of 9 patients post-chemotherapy. In summary, moderately intensive chemotherapy for HIV-1 related lymphoma in the context of continuous ART did not have a prolonged impact on HIV-1 persistence. © 2014 Cillo et al

Gold/Silica biochips: applications to Surface Plasmon Resonance and fluorescence quenching

We report Gold/Silica biochips for low cost biosensor devices. Firstly, the study of biochemical interactions on silica by means of Surface Plasmon Resonance (SPR) is presented. Secondly, Gold/Silica biochips are employed to reduce the strong quenching that occurs when a fluorophore is close to the gold surface. Furthermore, the control of the Silica-like thickness allows optimizing the distance between the metallic surface and the fluorophore in order to enhance the fluorescent signal. These results represent the first steps towards highly sensitive, specific and low cost biosensors based, for example, on Surface Plasmon Coupled Emission (SPCE) techniques

Genomics meets HIV-1

Genomics is now a core element in the effort to develop a vaccine against HIV-1. Thanks to unprecedented progress in high-throughput genotyping and sequencing, in knowledge about genetic variation in humans, and in evolutionary genomics, it is finally possible to systematically search the genome for common genetic variants that influence the human response to HIV-1. The identification of such variants would help to determine which aspects of the response to the virus are the most promising targets for intervention. However, a key obstacle to progress remains the scarcity of appropriate human cohorts available for genomic research

Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development

RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function

Likely Role of APOBEC3G-Mediated G-to-A Mutations in HIV-1 Evolution and Drug Resistance

The role of APOBEC3 (A3) protein family members in inhibiting retrovirus infection and mobile element retrotransposition is well established. However, the evolutionary effects these restriction factors may have had on active retroviruses such as HIV-1 are less well understood. An HIV-1 variant that has been highly G-to-A mutated is unlikely to be transmitted due to accumulation of deleterious mutations. However, G-to-A mutated hA3G target sequences within which the mutations are the least deleterious are more likely to survive selection pressure. Thus, among hA3G targets in HIV-1, the ratio of nonsynonymous to synonymous changes will increase with virus generations, leaving a footprint of past activity. To study such footprints in HIV-1 evolution, we developed an in silico model based on calculated hA3G target probabilities derived from G-to-A mutation sequence contexts in the literature. We simulated G-to-A changes iteratively in independent sequential HIV-1 infections until a stop codon was introduced into any gene. In addition to our simulation results, we observed higher ratios of nonsynonymous to synonymous mutation at hA3G targets in extant HIV-1 genomes than in their putative ancestral genomes, compared to random controls, implying that moderate levels of A3G-mediated G-to-A mutation have been a factor in HIV-1 evolution. Results from in vitro passaging experiments of HIV-1 modified to be highly susceptible to hA3G mutagenesis verified our simulation accuracy. We also used our simulation to examine the possible role of A3G-induced mutations in the origin of drug resistance. We found that hA3G activity could have been responsible for only a small increase in mutations at known drug resistance sites and propose that concerns for increased resistance to other antiviral drugs should not prevent Vif from being considered a suitable target for development of new drugs

A putative antiviral role of plant cytidine deaminases

[EN] Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade. Host cytidine deaminases (e.g., APOBEC3 proteins) edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis. By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity. To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation. The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases (AtCDAs), raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV). Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thaliana AtCDA genes in Nicotiana bigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions. Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes. This and control plants were then infected with CaMV. Virus accumulation and mutational spectra where characterized in both types of plants. Results: We have shown that the A. thaliana AtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated. Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.This work was supported by the former Spanish Ministerio de Ciencia e Innovación-FEDER grant BFU2009-06993 to SFE. JMC was supported by the CSIC JAE-doc program/Fondo Social Europeo. AG-P was supported by a grant for Scientific and Technical Activities and by grant P10-CVI-65651, both from Junta de Andalucía.Martín, S.; Cuevas, J.; Grande-Perez, A.; Elena Fito, SF. (2017). A putative antiviral role of plant cytidine deaminases. F1000Research. 1-14. https://doi.org/10.12688/f1000research.11111.2S11