scholarly journals Lack of Mutational Hot Spots during Decitabine-Mediated HIV-1 Mutagenesis

2015 ◽  
Vol 59 (11) ◽  
pp. 6834-6843 ◽  
Author(s):  
Jonathan M. O. Rawson ◽  
Sean R. Landman ◽  
Cavan S. Reilly ◽  
Laurent Bonnac ◽  
Steven E. Patterson ◽  
...  
Keyword(s):  
Hot Spots ◽  
Mutation Frequency ◽  
High Throughput Sequencing ◽  
Fold Increase ◽  
Lethal Mutagenesis ◽  
Immunodeficiency Virus ◽  
Mutagenic Effects ◽  
First Time ◽  
Hiv 1

ABSTRACTDecitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity.

scholarly journals 5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1

2009 ◽  
Vol 83 (22) ◽  
pp. 11950-11958 ◽  
Author(s):  
Michael J. Dapp ◽  
Christine L. Clouser ◽  
Steven Patterson ◽  
Louis M. Mansky
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcription ◽  
Mutation Frequency ◽  
Late Phase ◽  
Viral Dna ◽  
Lethal Mutagenesis ◽  
Immunodeficiency Virus ◽  
Antiviral Mechanism ◽  
Hiv 1

ABSTRACT Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription.

scholarly journals Antihuman Immunodeficiency Virus Type 1 (HIV-1) Activity of Rare Earth Metal Complexes of 4-Hydroxycoumarins in Cell Culture

2006 ◽  
Vol 2006 ◽  
pp. 1-7 ◽  
Author(s):  
Ilia Manolov ◽  
Sevda Raleva ◽  
Petya Genova ◽  
Alexey Savov ◽  
Liliana Froloshka ◽  
...  
Keyword(s):  
Viral Replication ◽  
Earth Metal ◽  
H Nmr ◽  
Immunodeficiency Virus ◽  
Physicochemical Methods ◽  
Anti Hiv Agents ◽  
First Time ◽  
Anti Hiv ◽  
Hiv 1

The cerium Ce(III), lanthanum La(III), and neodymium Nd(III) complexes with 4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin) (W) and 3,3′-benzylidenebis[4-hydroxycoumarin] (1) were synthesized and studied for the first time for cytotoxicity (on MT-2 cells) and as anti-HIV agents under acute and chronic infection. The complexes were characterized by different physicochemical methods: mass spectrometry, ¹H NMR, ¹³C NMR, and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligands. Anti-HIV effect of the complexes/ligands was measured in MT-2 cells by microtiter infection assay. Detection of endogenous reverse transcriptase (RT) activity and RT processivity by PCR indicative for proviral DNA synthesis demonstrated that anti-HIV activity has not been linked to early stages of viral replication. No effect on late steps of viral replication has been found using cells chronically producing HIV-1LAIvirus. La(W) demonstrated anti-HIV activity (IC50=21.4μM) close to maximal nontoxic concentration. Nd(W), Ce(1), and Nd(1) demonstrated limited anti-HIV potency, so none of the complexes seems appropriate to be used in clinic. Further targeting of HIV-1 inhibition by La(W) is under progress.

scholarly journals Patient-Specific Cytotoxic T-Lymphocyte Cross-Recognition of Naturally Occurring Variants of a Human Immunodeficiency Virus Type 1 (HIV-1) p24gagEpitope by HIV-1-Infected Children

2001 ◽  
Vol 75 (10) ◽  
pp. 4941-4946 ◽  
Author(s):  
F. Buseyne ◽  
M.-L. Chaix ◽  
C. Rouzioux ◽  
S. Blanche ◽  
Y. Rivière
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Patient Specific ◽  
Interindividual Variations ◽  
Naturally Occurring ◽  
Immunodeficiency Virus ◽  
First Time ◽  
Hiv 1

ABSTRACT We tested seven human immunodeficiency virus-infected children for their cytotoxic T-lymphocyte (CTL) activities towards the p24 gag QASQEVKNW epitope and its nine variant sequences. Our data confirm that most, but not all, CTL responses are broadly cross-specific. For the first time, we show the high interpatient variability in cross-recognition of mutant CTL epitopes. These interindividual variations in the CTL response to the same epitope should be taken into account in the design and the evaluation of vaccines.

scholarly journals Correlation of Phenotypic Zidovudine Resistance with Mutational Patterns in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1: Interpretation of Established Mutations and Characterization of New Polymorphisms at Codons 208, 211, and 214

2003 ◽  
Vol 47 (1) ◽  
pp. 54-61 ◽  
Author(s):  
Martin Stürmer ◽  
Schlomo Staszewski ◽  
Hans-Wilhelm Doerr ◽  
Brendan Larder ◽  
Stuart Bloor ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fold Increase ◽  
Clinical Samples ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
M184v Mutation

ABSTRACT Zidovudine resistance (ZDV-R) is associated with classic genotypic changes at codons 41, 67, 70, 210, 215, and 219 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene as well as with the multinucleoside resistance (MNR) complexes (Q151M MNR complex; 6-bp insertion/A62V complex). In addition, enhanced resistance to ZDV in the context of the classic ZDV mutations plus the M184V mutation has been associated with additional mutations at positions 208, 211, 214, and 333. In this study we investigated phenotypic ZDV-R determined by a recombinant virus assay (Antivirogram; Virco) in 223 clinical samples in relation to the above genotypic changes. 150 out of 223 clinical samples had the M184V mutation. Phenotypic ZDV-R ranged from 0.3- to 5,338-fold. Sixteen samples (15 with high ZDV-R ranging from 90- to 3,571-fold) contained MNR-associated patterns. Analysis of classic mutational patterns broadly demonstrated increasing ZDV-R with increasing number of ZDV mutations. A comparable correlation was obtained when ZDV-R was analyzed only relative to the T215Y/F mutation. Site-directed mutagenesis experiments investigating the influence of the additional mutations H208Y, R211K, and L214F on ZDV-R resulted in a 7.4- or 21-fold increase in ZDV-R when the R211K/L214F or H208Y/R211K/L214F mutations, respectively, were added to a highly ZDV-R virus. In the clinical sample data set we analyzed, the combination of R211K/L214F appeared most frequently. The H208Y change was detected only in highly ZDV-R viruses, whereas the G333E/D change was distributed equally. All changes were independent of the M184V mutation. A 2.4- or 8-fold increase in ZDV-R was observed in the clinical samples with high ZDV-R containing the R211K/L214F or H208Y/R211K/L214F mutations, respectively. We have shown that the combination of the additional mutations H208Y, R211K, and L214F in HIV-1 RT may influence ZDV-R and should be considered when assessing ZDV-R.

scholarly journals Human Immunodeficiency Virus Type 1 Recombination: Rate, Fidelity, and Putative Hot Spots

2002 ◽  
Vol 76 (22) ◽  
pp. 11273-11282 ◽  
Author(s):  
Jianling Zhuang ◽  
Amanda E. Jetzt ◽  
Guoli Sun ◽  
Hong Yu ◽  
George Klarmann ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Sequencing ◽  
Human Immunodeficiency Virus Type ◽  
Hot Spots ◽  
Hot Spot ◽  
High Rate ◽  
Vector System ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

scholarly journals Mouse-Human Heterokaryons Support Efficient Human Immunodeficiency Virus Type 1 Assembly

2001 ◽  
Vol 75 (7) ◽  
pp. 3141-3151 ◽  
Author(s):  
Roberto Mariani ◽  
Beth A. Rasala ◽  
Gabriel Rutter ◽  
Klaus Wiegers ◽  
Stephanie M. Brandt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Fold Increase ◽  
Human Cells ◽  
Virus Release ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

scholarly journals CXCR4-Dependent Infection of CD8+, but Not CD4+, Lymphocytes by a Primary Human Immunodeficiency Virus Type 1 Isolate

2004 ◽  
Vol 78 (22) ◽  
pp. 12288-12296 ◽  
Author(s):  
Bouchra Zerhouni ◽  
Julie A. E. Nelson ◽  
Kunal Saha
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Specific Antagonist ◽  
T Cell Lines ◽  
First Time ◽  
Hiv 1

ABSTRACT We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8+ lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4+ cells. In the present study, we investigated the role of CXCR4 in the infection of CD4+ and CD8+ cells by this primary isolate. The expression of CXCR4 was down modulated in CD8+ lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8+ lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4+ lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4+ CXCR4+ T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4+ cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4+ cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8+ lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4+ and CD8+ lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.

scholarly journals Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs

2016 ◽  
Vol 198 (20) ◽  
pp. 2776-2783 ◽  
Author(s):  
Jocelyn Ang ◽  
Lisa Yun Song ◽  
Sara D'Souza ◽  
Irene L. Hong ◽  
Rohan Luhar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Hot Spots ◽  
Mutation Frequency ◽  
Fold Increase ◽  
Content Type ◽  
Thymidine Analog ◽  
Base Analog ◽  
Cytidine Analogs ◽  
Mutagenic Effects

ABSTRACTWe tested pairwise combinations of classical base analog mutagens inEscherichia colito study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10−8) in therpoBgene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools.IMPORTANCEAlthough mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.

scholarly journals 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

2016 ◽  
Vol 60 (4) ◽  
pp. 2318-2325 ◽  
Author(s):  
Jonathan M. O. Rawson ◽  
Michele B. Daly ◽  
Jiashu Xie ◽  
Christine L. Clouser ◽  
Sean R. Landman ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lethal Mutagenesis ◽  
Immunodeficiency Virus ◽  
Liquid Chromatography Tandem Mass ◽  
Hiv 1 ◽  
Deoxycytidine Triphosphate ◽  
Primary Form ◽  
In Cells

ABSTRACT5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2′-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography–tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

scholarly journals N4-Acyl-Modified D-2′,3′-Dideoxy-5-Fluorocytidine Nucleoside Analogues with Improved Antiviral Activity

2003 ◽  
Vol 14 (2) ◽  
pp. 81-90 ◽  
Author(s):  
Junxing Shi ◽  
Judy S Mathew ◽  
Phillip M Tharnish ◽  
Suguna Rachakonda ◽  
S Balakrishna Pai ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Parent Compound ◽  
Direct Interaction ◽  
Fold Increase ◽  
B Virus ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

A series of 2,3-dideoxy (D2) and 2,3-didehydro-2,3-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4- p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4- p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4- p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5′-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5′-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.

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