scholarly journals Triplet Analysis That Identifies Unpaired Regions of Functional RNAs

2011 ◽  
Vol 2011 ◽  
pp. 1-6
Author(s):  
Junji Kawakami ◽  
Yoshie Yamaguchi ◽  
Naoki Sugimoto
Keyword(s):  
Consensus Sequence ◽  
Structural Features ◽  
Analysis Method ◽  
Primary Sequence ◽  
Rna Sequences ◽  
Unpaired Region ◽  
Novel Method ◽  
Functional Rnas ◽  
Hiv 1

We developed a novel method for analyzing RNA sequences, deemed triplet analysis, and applied the method in anin vitroRNA selection experiment in which HIV-1 Tat was the target. Aptamers are nucleic acids that bind a desired target (bait), and to date, many aptamers have been identified byin vitroselection from enough concentrated libraries in which many RNAs had an obvious consensus primary sequence after sufficient cycles of the selection. Therefore, the higher-order structural features of the aptamers that are indispensable for interaction with the bait must be determined by additional investigation of the aptamers. In contrast, our triplet analysis enabled us to extract important information on functional primary and secondary structure from minimally concentrated RNA libraries. As a result, by using our method, an important unpaired region that is similar to the bulge of TAR was readily predicted from a partially concentrated library in which no consensus sequence was revealed by a conventional sequence analysis. Moreover, our analysis method may be used to assess a variety of structural motifs with desired function.

Synthesis and Evaluation of 3-(1,3-dioxoisoindolin-2-yl)-N-substituted Phenyl Benzamide Analogues as HIV Integrase Strand Transfer Inhibitors

2019 ◽  
Vol 17 (2) ◽  
pp. 105-114
Author(s):  
Pankaj Wadhwa ◽  
Priti Jain ◽  
Arpit Patel ◽  
Shantanu Shinde ◽  
Hemant R. Jadhav
Keyword(s):  
Structural Features ◽  
Docking Studies ◽  
Significant Inhibition ◽  
Strand Transfer ◽  
Hiv Integrase ◽  
In Silico Docking ◽  
Immunodeficiency Virus ◽  
Hiv 1

<P>Background: A series of novel 3-(1,3-dioxoisoindolin-2-yl)-N-substituted phenyl benzamide derivatives was synthesized and tested in vitro against human immunodeficiency virus type-1 Integrase (HIV-1 IN). Methods: Out of the 18 analogues, six (compounds 16c, 16h, 16i, 16m, 16n and 16r) showed significant inhibition of strand transfer by HIV-1 integrase. For these six compounds. IC50 was below 5.0 µM. In silico docking studies revealed that the presence of 2-phenyl isoindoline-1,3-dione motif was essential as it was found to interact with active site magnesium. Results: To further confirm the results, cell-based HIV-1 and HIV-2 inhibitory assay was carried out. Conclusion: These compounds possess structural features not seen in previously reported HIV-1 integrase inhibitors and thus can help further optimization of anti-HIV-1 integrase activity.</P>

scholarly journals In SilicoandIn VitroComparison of HIV-1 Subtypes B and CRF02_AG Integrases Susceptibility to Integrase Strand Transfer Inhibitors

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Xiaoju Ni ◽  
Safwat Abdel-Azeim ◽  
Elodie Laine ◽  
Rohit Arora ◽  
Osamuede Osemwota ◽  
...  
Keyword(s):  
Structural Features ◽  
Strand Transfer ◽  
Binding Modes ◽  
Medical Treatments ◽  
Sequence Variations ◽  
Transfer Inhibitor ◽  
Dna Substrate ◽  
Integrase Strand Transfer Inhibitor ◽  
Hiv 1

Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors—raltegravir (RAL), elvitegravir (ELV) and L731,988—were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Ourin silicoandin vitrostudies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.

scholarly journals Novel Protease Inhibitors (PIs) Containing Macrocyclic Components and 3(R),3a(S),6a(R)-bis-Tetrahydrofuranylurethane That Are Potent against Multi-PI-Resistant HIV-1 Variants In Vitro

2010 ◽  
Vol 54 (8) ◽  
pp. 3460-3470 ◽  
Author(s):  
Yasushi Tojo ◽  
Yasuhiro Koh ◽  
Masayuki Amano ◽  
Manabu Aoki ◽  
Debananda Das ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Molecular Design ◽  
Antiviral Agent ◽  
Structural Features ◽  
Biological Properties ◽  
Van Der Waals Interactions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Hiv 1

ABSTRACT Natural products with macrocyclic structural features often display intriguing biological properties. Molecular design incorporating macrocycles may lead to molecules with unique protein-ligand interactions. We generated novel human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a macrocycle and bis-tetrahydrofuranylurethane. Four such compounds exerted potent activity against HIV-1LAI and had 50% effective concentrations (EC50s) of as low as 0.002 μM with minimal cytotoxicity. GRL-216 and GRL-286 blocked the replication of HIV-1NL4-3 variants selected by up to 5 μM saquinavir, ritonavir, nelfinavir, lopinavir, or atazanavir; they had EC50s of 0.020 to 0.046 μM and potent activities against six multi-PI-resistant clinical HIV-1 (HIVmPIr ) variants with EC50s of 0.027 to 0.089 μM. GRL-216 and -286 also blocked HIV-1 protease dimerization as efficiently as darunavir. When HIV-1NL4-3 was selected by GRL-216, it replicated progressively more poorly and failed to replicate in the presence of >0.26 μM GRL-216, suggesting that the emergence of GRL-216-resistant HIV-1 variants is substantially delayed. At passage 50 with GRL-216 (the HIV isolate selected with GRL-216 at up to 0.16 μM [HIV216-0.16 μM]), HIV-1NL4-3 containing the L10I, L24I, M46L, V82I, and I84V mutations remained relatively sensitive to PIs, including darunavir, with the EC50s being 3- to 8-fold-greater than the EC50 of each drug for HIV-1NL4-3. Interestingly, HIV216-0.16 μM had 10-fold increased sensitivity to tipranavir. Analysis of the protein-ligand X-ray structures of GRL-216 revealed that the macrocycle occupied a greater volume of the binding cavity of protease and formed greater van der Waals interactions with V82 and I84 than darunavir. The present data warrant the further development of GRL-216 as a potential antiviral agent for treating individuals harboring wild-type and/or HIVmPIr .

scholarly journals Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1874
Author(s):  
Alice Sosic ◽  
Giulia Olivato ◽  
Caterina Carraro ◽  
Richard Göttlich ◽  
Dan Fabris ◽  
...  
Keyword(s):  
Rna Sequences ◽  
Protein Inhibition ◽  
Rna Targeting ◽  
Activation Response ◽  
Tar Rna ◽  
Novel Strategy ◽  
Bulge Loop ◽  
Targeting Strategy ◽  
Hiv 1

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem–bulge–loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs’ recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

scholarly journals An Expanded Class of Histidine-Accepting Viral tRNA-like Structures

2020 ◽  
Author(s):  
Conner J. Langeberg ◽  
Madeline E. Sherlock ◽  
Andrea MacFadden ◽  
Jeffrey S. Kieft
Keyword(s):  
Consensus Sequence ◽  
Structure Model ◽  
Host Proteins ◽  
Secondary Structure Model ◽  
Rna Sequences ◽  
Chemical Probing ◽  
Bioinformatic Approaches ◽  
Tertiary Interaction ◽  
Rna Elements

ABSTRACTStructured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3′ termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate several processes important for infection, to include replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to verify a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structures and functions in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

scholarly journals Low recombination activity of R region located at both ends of the HIV-1 genome.

2012 ◽  
Vol 59 (4) ◽  
Author(s):  
Anna Urbanowicz ◽  
Anna Kurzyńska-Kokorniak ◽  
Anna Jankowska ◽  
Magdalena Alejska ◽  
Marek Figlerowicz
Keyword(s):  
Rna Viruses ◽  
Brome Mosaic Virus ◽  
Template Switching ◽  
Strand Transfer ◽  
Recombination Activity ◽  
Rna Sequences ◽  
R Region ◽  
Hiv 1

Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus--BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses.

scholarly journals Genetic Variation and Susceptibilities to Protease Inhibitors among Subtype B and F Isolates in Brazil

1999 ◽  
Vol 43 (2) ◽  
pp. 253-258 ◽  
Author(s):  
Amilcar Tanuri ◽  
Ana C. P. Vicente ◽  
Koko Otsuki ◽  
Carlos A. Ramos ◽  
Orlando C. Ferreira ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Consensus Sequence ◽  
Antiretroviral Drugs ◽  
Protease Gene ◽  
Subtype B ◽  
Hiv 1 ◽  
The U.S

ABSTRACT The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow thegag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilianprt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

scholarly journals Armored RNA Technology for Production of Ribonuclease-Resistant Viral RNA Controls and Standards

1998 ◽  
Vol 36 (12) ◽  
pp. 3590-3594 ◽  
Author(s):  
Brittan L. Pasloske ◽  
Cindy R. Walkerpeach ◽  
R. Dawn Obermoeller ◽  
Matthew Winkler ◽  
Dwight B. DuBois
Keyword(s):  
Escherichia Coli ◽  
Coat Protein ◽  
Consensus Sequence ◽  
Expression Plasmid ◽  
Rna Sequences ◽  
Immunodeficiency Virus ◽  
Rnase Digestion ◽  
Armored Rna ◽  
Hiv 1

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. “Armored RNA” is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at −20°C or for 60 days at 4°C. Additionally, Armored RNA preparations are homogeneous and noninfectious.

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro

1992 ◽  
Vol 12 (9) ◽  
pp. 3699-3705
Author(s):  
A Valsamakis ◽  
N Schek ◽  
J C Alwine
Keyword(s):  
Human Immunodeficiency Virus ◽  
Consensus Sequence ◽  
In Vivo Studies ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Substitution Mutations ◽  
Hiv 1

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.

scholarly journals Inhibition of HIV-1 Replication by a Bis-Thiadiazolbenzene-1,2-Diamine That Chelates Zinc Ions from Retroviral Nucleocapsid Zinc Fingers

2010 ◽  
Vol 54 (4) ◽  
pp. 1461-1468 ◽  
Author(s):  
Christophe Pannecouque ◽  
Beata Szafarowicz ◽  
Natalia Volkova ◽  
Vasiliy Bakulev ◽  
Wim Dehaen ◽  
...  
Keyword(s):  
Viral Replication ◽  
Wide Spectrum ◽  
Zinc Fingers ◽  
Sulfhydryl Group ◽  
Structural Features ◽  
Acyl Transfer ◽  
Mechanistic Study ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 (NCp7) protein holds two highly conserved “CCHC” zinc finger domains that are required for several phases of viral replication. Basic residues flank the zinc fingers, and both determinants are required for high-affinity binding to RNA. Several compounds were previously found to target NCp7 by reacting with the sulfhydryl group of cysteine residues from the zinc fingers. Here, we have identified an N,N′-bis(1,2,3-thiadiazol-5-yl)benzene-1,2-diamine (NV038) that efficiently blocks the replication of a wide spectrum of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) strains. Time-of-addition experiments indicate that NV038 interferes with a step of the viral replication cycle following the viral entry but preceding or coinciding with the early reverse transcription reaction, pointing toward an interaction with the nucleocapsid protein p7. In fact, in vitro, NV038 efficiently depletes zinc from NCp7, which is paralleled by the inhibition of the NCp7-induced destabilization of cTAR (complementary DNA sequence of TAR). A chemical model suggests that the two carbonyl oxygens of the esters in this compound are involved in the chelation of the Zn2+ ion. This compound thus acts via a different mechanism than the previously reported zinc ejectors, as its structural features do not allow an acyl transfer to Cys or a thiol-disulfide interchange. This new lead and the mechanistic study presented provide insight into the design of a future generation of anti-NCp7 compounds.

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