PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA

BACKGROUND: The HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNA through interaction with the Rev response element (RRE) by means of an arginine rich motif that is similar to the one found in Tat. Since Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its arginine rich motif, we investigated whether the Rev protein could act as a substrate for this enzyme. RESULTS: Here, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner. CONCLUSION: These findings indicate that PRMT6 severely impairs the function of HIV-1 Rev

Benefits of an educational program for journalists on media coverage of HIV/AIDS in developing countries

Benefits of an educational program for journalists on medi

A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1

Molecular characterization of the development of the K65R and M184V drug resistance mutations in Subtype C HIV-1s

Background: We have shown that the K65R mutation is selected more rapidly in subtype C than in subtype B HIV-1 isolates in both cell culture and clinical studies. Biochemical comparisons between subtype B and C-derived reverse transcriptase (RT) enzymes revealed similar molecular characteristics that do not explain the more rapid selection of K65R with subtype C viruses. This study attempts to establish the mechanistic basis for the difference. Methods: Recombinant subtype C and B HIV-1 RT enzymes were expressed and purified in E. coli. Gel-based nucleotide extension assays were used to study DNA synthesis from various natural and synthetic DNA and RNA templates that spanned regions of the pol gene responsible for the K65R and M184V mutations. Cell based experiments were performed using MT2 cells infected with mutated subtype B HIV-1 pNL4-3 viruses. Results: The propensity for the more rapid selection of K65R with subtype C enzymes is due to the mechanism of DNA synthesis from a subtype C template. The use of templates containing the 64, 65 and 66 codons of the pol gene led to different patterns of DNA synthesis. When subtype C RT was employed to synthesize DNA from subtype C templates, preferential pausing was seen at the nucleotide position responsible for the AAG to AGG mutation on codon 65 which gives rise to K65R. In contrast, the use of subtype B RT together with a subtype B template reveals a different pattern of DNA synthesis. When subtype B RT was employed with a subtype C template, DNA synthesis stopped at the exact nucleotide position responsible for K65R. This phenomenon was not observed when subtype C RT was used with a subtype B template. A similar method was employed to investigate if differences exist in the appearance of M184V between subtypes. The results suggest that M184V is not favoured due to its coding sequence and that the propensity for the development of M184V remains the same in subtype B and C HIV. In cell culture, K65R was detected faster in subtype B that has been mutated to include the 64/65 codons of subtype C, when compared to wild-type subtype B HIV. Conclusions: The more rapid emergence of K65R but not M184V in subtype C RT appears to be based on the pol gene coding sequence. These results urge for the analysis of resistance mechanisms to be studied in all HIV subtypes separately and have clinical relevance in regard to the management of subtype C infections

New pyridobenzoxazepine derivatives derived from 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine (JL13): chemical synthesis and pharmacological evaluation

A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized in order to explore two main parameters related to the distal basic nitrogen. These compounds were tested for their affinity for dopamine D2L, D4, serotonin 5-HT1A, 5-HT2A and adrenergic 2A receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1) and other diarylazepine derivatives. In terms of multi-receptor target strategy, 2 and 5 present the most promising in vitro binding profile. Bulky, polar and more flexible side chains are not favourable in this context. 2 and 5 were tested in adult rats to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D1 and D4 receptors in nucleus accumbens and caudate putamen, and D2 receptors in medial prefrontal cortex and hippocampus while 5 significantly increased D2 and D4 receptors in nucleus accumbens. In addition, 2 increased 5-HT1A and decreased 5-HT2A receptors in cerebral cortex. In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further development of 2 as a novel second-generation antipsychotic agent.Composés antipsychotique

ARC: An Open Web-Platform for Request/Supply Matching for a Prioritized and Controlled COVID-19 Response

ISSN:2296-256