Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mutations in the human immunodeficiency virus type 1 polypurine tract (PPT) reduce the rate of PPT cleavage and plus-strand DNA synthesis.

ABSTRACT: Previously, we analyzed the effects of point mutations in the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT) and found that some mutations affected both titer and cleavage specificity. We used HIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay to measure differences in the relative rates of PPT processing and utilization. The relative rates were measured by determining which of the two PPTs in the vector is used to synthesize viral DNA. The results indicate that mutations that have subtle effects on titer and cleavage specificity can have dramatic effects on rates of PPT generation and utilization.

SUBMITTER: McWilliams MJ

PROVIDER: S-EPMC2346737 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Mutations in the human immunodeficiency virus type 1 polypurine tract (PPT) reduce the rate of PPT cleavage and plus-strand DNA synthesis.

McWilliams M J MJ Julias J G JG Hughes S H SH

Journal of virology 20080305 10

Previously, we analyzed the effects of point mutations in the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT) and found that some mutations affected both titer and cleavage specificity. We used HIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay to measure differences in the relative rates of PPT processing and utilization. The relative rates were measured by determining which of the two PPTs in the vector is used to synthesize ...[more]

PMID: 18321979

Similar Datasets

Sequence and structural determinants required for priming of plus-strand DNA synthesis by the human immunodeficiency virus type 1 polypurine tract.

Project description:At the 3' end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3' end of the PPT. In addition, we find that a hybrid consisting of a 15- or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5' end of the PPT have no effect on primer function, whereas the identity of bases at the 3' end is crucial. A primer containing only the 6 G residues from the 3' end of the wild-type PPT sequence and 9 bases of random sequence at the 5' end functions like a wild-type PPT. A short hybrid having a similar helical structure but a primary sequence different from that of the PPT is cleaved imprecisely, resulting in initiation of synthesis at multiple sites; however, total primer extension is close to the wild-type level. We conclude that helical structure as well as the presence of particular bases at the 3' end of the PPT is essential for PPT function.

| S-EPMC190486 | biostudies-other

Specific mutations in the HIV-1 G-tract of the 3'-polypurine tract cause resistance to integrase strand transfer inhibitors.

Project description:BackgroundIn vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3'-polypurine tract (3'-PPT). A clinical study also reported that mutations in the 3'-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3'-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs.ObjectivesTo identify the specific mutation(s) in the G-tract of the 3'-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs.MethodsThe infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells.ResultsA single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3'-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or 'T' at the second position and 'T' at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose-response curves, consistent with a non-competitive mechanism of inhibition.ConclusionsMutations at the second and fifth positions in the G-tract of the 3'-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.

| S-EPMC8865006 | biostudies-literature

Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis.

Project description:HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3'-end. The integrity of the 3'-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.

| S-EPMC4344514 | biostudies-literature

SJP-L-5 inhibits HIV-1 polypurine tract primed plus-strand DNA elongation, indicating viral DNA synthesis initiation at multiple sites under drug pressure.

Project description:In a previous study the small molecule SJP-L-5 that inhibits HIV replication, has been shown to block uncoating of the viral capsid. Continued study showed that SJP-L-5 might hinder HIV capsid uncoating by blocking the completion of reverse transcription. However, to date, the mechanism has not been fully elucidated. Here, the effects of SJP-L-5 for reverse transcription were explored via quantitative PCR, DIG-labelled ELISA, fluorescent resonance energy transfer, and Southern blot assays. We also analyzed the resistance profile of this compound against reverse transcriptase. Our results show that SJP-L-5 preferentially inhibits PPT primed plus-strand DNA synthesis (EC50 = 13.4 ± 3.0 μM) over RNA primed minus-strand DNA synthesis (EC50 > 3,646 μM), resulting in formation of five segmented plus-strand DNA and loss of HIV DNA flap, suggesting failure of both nuclear import and integration. Moreover, resistance study evidenced that SJP-L-5 requires the amino acid residues Val108 and Tyr181 to exert an inhibitory effect. These results indicate SJP-L-5 as a new non-nucleoside reverse transcriptase inhibitor that inhibits HIV-1 polypurine tract primed plus-strand DNA synthesis, initiating HIV-1 down-stream plus-strand DNA synthesis at multiple sites under drug pressure.

| S-EPMC5803243 | biostudies-literature

Specific recognition and cleavage of the plus-strand primer by reverse transcriptase.

Project description:Reverse transcriptases (RTs) of retroviruses and long terminal repeat (LTR)-retrotransposons possess DNA polymerase and RNase H activities. During reverse transcription these activities are necessary for the programmed sequence of events that include template switching and primer processing. Integrase then inserts the completed cDNA into the genome of the host cell. The RT of the LTR-retrotransposon Tf1 was subjected to random mutagenesis, and the resulting transposons were screened with genetic assays to test which mutations reduced reverse transcription and which inhibited integration. We identified a cluster of mutations in the RNase H domain of RT that were surprising because they blocked integration without reducing cDNA levels. The results of immunoblots demonstrated that these mutations did not reduce levels of RT or integrase. DNA blots showed that the mutations did not lower the amounts of full-length cDNA. The sequences of the 3' ends of the cDNA revealed that mutations within the cluster in RNase H specifically reduced the removal of the polypurine tract (PPT) primer from the ends of the cDNA. These results indicate that primer removal is not a necessary component of reverse transcription. The residues mutated in Tf1 RNase H are conserved in human immunodeficiency virus type 1 and make direct contact with DNA opposite the PPT. Thus, our results identify a conserved element in RT that contacts the PPT and is specifically required for PPT removal.

| S-EPMC1287563 | biostudies-literature

Identification of a sequence element immediately upstream of the polypurine tract that is essential for replication of simian immunodeficiency virus.

Project description:A short stretch of T-rich sequences immediately upstream of the polypurine tract (PPT) is highly conserved in the proviral genomes of human and simian immunodeficiency viruses (HIV and SIV). To investigate whether this 'U-box' influences SIVmac239 replication, we analyzed the properties of mutants with changes in this region of the viral genome. All mutants were either retarded in their growth (up to one month delay) or did not replicate detectably in CEMx174 cells. When U-box mutants did replicate detectably, compensatory changes were consistently observed in the viral genome. The most common compensatory change was the acquisition of thymidines immediately upstream of the PPT, but marked expansion in the length of the PPT was also observed. U-box mutants produced transiently by transfection were severely impaired in their ability to produce reverse transcripts in infectivity assays. Analysis of transiently produced mutant virus revealed no defect in RNA packaging or virus assembly. These results identify a new structural element important for an early step in the viral life cycle that includes reverse transcription.

| S-EPMC1170712 | biostudies-other

Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.

Project description:HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

| S-EPMC5766924 | biostudies-literature

Human immunodeficiency virus type 1 protease-correlated cleavage site mutations enhance inhibitor resistance.

Project description:Drug resistance is an important cause of antiretroviral therapy failure in human immunodeficiency virus (HIV)-infected patients. Mutations in the protease render the virus resistant to protease inhibitors (PIs). Gag cleavage sites also mutate, sometimes correlating with resistance mutations in the protease, but their contribution to resistance has not been systematically analyzed. The present study examines mutations in Gag cleavage sites that associate with protease mutations and the impact of these associations on drug susceptibilities. Significant associations were observed between mutations in the nucleocapsid-p1 (NC-p1) and p1-p6 cleavage sites and various PI resistance-associated mutations in the protease. Several patterns were frequently observed, including mutations in the NC-p1 cleavage site in combination with I50L, V82A, and I84V within the protease and mutations within the p1-p6 cleavage site in combination with D30N, I50V, and I84V within the protease. For most patterns, viruses with mutations both in the protease and in either cleavage site were significantly less susceptible to specific PIs than viruses with mutations in the protease alone. Altered PI resistance in HIV-1 was found to be associated with the presence of Gag cleavage site mutations. These studies suggest that associated cleavage site mutations may contribute to PI susceptibility in highly specific ways depending on the particular combinations of mutations and inhibitors. Thus, cleavage site mutations should be considered when assessing the level of PI resistance.

| S-EPMC2772784 | biostudies-literature

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

Project description:HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342-351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342-350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

| S-EPMC7831940 | biostudies-literature

NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling.

Project description:Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.

| S-EPMC2722892 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data