SUMMARY While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild type but not escape mutant virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.
The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1-4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5-7). For example, the γ-retroviruses favor integration near transcription start sites, whereas lentiviruses favor integration within transcription units. These observations have suggested that different cellularbinding partners of retroviral integrases are likely to be responsible for integration target-site selection. However, to date, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes (8-14). Cellular cofactors of other retroviral genera are currently unknown.The molecular mechanisms of γ-retroviral murine leukemia virus (MLV) integration are of particular significance because MLV-based vectors are used for human gene therapy. In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. 15-18). The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.In this report, we have identified the bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as the cellularbinding partners of MLV IN and demonstrate their significance for stimulating and targeting MLV integration at transcription start sites. (Table 1, Table S1, and Fig. S1). Of these, Brd4 and Brd3 were the top hits in NIH 3T3 and Sup-T1 cells, respectively. Differential pull-down levels of these proteins (Table 1) could be attributable to the varying expression le...
Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.
It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm.
Background:The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs). Results: The A128T substitution does not significantly alter ALLINI IC 50 values for IN-LEDGF/p75 binding but confers marked resistance to ALLINI-induced aberrant integrase multimerization. Conclusion: Allosteric perturbation of HIV-1 integrase multimerization underlies ALLINI antiviral activity. Significance: Our findings underscore the mechanism of ALLINI action and will facilitate development of second-generation compounds.
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