scholarly journals Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

scholarly journals Antiviral Activity of Bictegravir and Cabotegravir against Integrase Inhibitor-Resistant SIVmac239 and HIV-1

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Said A. Hassounah ◽  
Ahmad Alikhani ◽  
Maureen Oliveira ◽  
Simrat Bharaj ◽  
Ruxandra-Ilinca Ibanescu ◽  
...  
Keyword(s):  
Treatment Strategies ◽  
Fold Increase ◽  
Integrase Inhibitor ◽  
Antiretroviral Drugs ◽  
Strand Transfer ◽  
Single Cycle ◽  
Genetic Barrier ◽  
Hiv 1

ABSTRACT Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.

Effect of Antibody to HIV-1 Tat Protein on Viral Replication in Vitro and Progression of HIV-1 Disease in Vivo

1995 ◽  
Vol 10 (4) ◽  
pp. 408-416 ◽  
Author(s):  
M C Re ◽  
G Furlini ◽  
M Vignoli ◽  
E Ramazzotti ◽  
G Roderigo ◽  
...  
Keyword(s):  
Viral Replication ◽  
Tat Protein ◽  
Hiv 1

scholarly journals In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

RNA Biology ◽  
2011 ◽  
Vol 8 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Sébastien Lainé ◽  
Robert J. Scarborough ◽  
Dominique Lévesque ◽  
Ludovic Didierlaurent ◽  
Kaitlin J. Soye ◽  
...  
Keyword(s):  
Viral Replication ◽  
Hiv 1

scholarly journals In Vivo Evidence for Instability of Episomal Human Immunodeficiency Virus Type 1 cDNA

2005 ◽  
Vol 79 (8) ◽  
pp. 5203-5210 ◽  
Author(s):  
Mark Sharkey ◽  
Karine Triques ◽  
Daniel R. Kuritzkes ◽  
Mario Stevenson
Keyword(s):  
Viral Replication ◽  
In Vitro Experiments ◽  
Plasma Viremia ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.

scholarly journals Discordance between Frequency of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon-Producing CD4+ T Cells and HIV-1-Specific Lymphoproliferation in HIV-1-Infected Subjects with Active Viral Replication

2002 ◽  
Vol 76 (12) ◽  
pp. 5925-5936 ◽  
Author(s):  
B. E. Palmer ◽  
E. Boritz ◽  
N. Blyveis ◽  
C. C. Wilson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Replication ◽  
Th Cell ◽  
Immunodeficiency Virus ◽  
Active Viral Replication ◽  
Hiv 1

ABSTRACT One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4+ T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4+ Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4+ T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4+ T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4+ T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4+ T-cell proliferation.

scholarly journals Promiscuous targeting of cellular proteins by Vpr drives massive proteomic remodelling in HIV-1 infection

2018 ◽  
Author(s):  
Edward JD Greenwood ◽  
James C Williamson ◽  
Agata Sienkiewicz ◽  
Adi Naamati ◽  
Nicholas J Matheson ◽  
...  
Keyword(s):  
Viral Replication ◽  
Biological Significance ◽  
Cellular Proteins ◽  
M Cell ◽  
Cellular Targets ◽  
Host Restriction ◽  
Cellular Phenotypes ◽  
Hiv 1

AbstractHIV-1 encodes four ‘accessory proteins’ (Vif, Vpr, Vpu and Nef), dispensable for viral replication in vitro, but essential for viral pathogenesis in vivo. Well characterised cellular targets have been associated with Vif, Vpu and Nef, which counteract host restriction and promote viral replication. Conversely, whilst several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary, unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes, and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes, and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.

scholarly journals Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase

2019 ◽  
Vol 294 (20) ◽  
pp. 8286-8295 ◽  
Author(s):  
Clémence Richetta ◽  
Sylvain Thierry ◽  
Eloise Thierry ◽  
Paul Lesbats ◽  
Delphine Lapaillerie ◽  
...  
Keyword(s):  
Viral Replication ◽  
Long Terminal Repeat ◽  
Terminal Repeat ◽  
Host Genome ◽  
End Joining ◽  
Viral Dna ◽  
Retroviral Integrase ◽  
Hiv 1

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3′-processed–like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3′-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

A rapid and efficient system for screening HIV-1 Pol mRNA-specific ribozymes

1997 ◽  
Vol 43 (1) ◽  
pp. 92-96
Author(s):  
A. Ramezani ◽  
W. Marhin ◽  
M. Weerasinghe ◽  
S. Joshi
Keyword(s):  
Reverse Transcriptase ◽  
Hammerhead Ribozyme ◽  
Rapid Screening ◽  
Coding Region ◽  
Efficient System ◽  
Target Sites ◽  
Hiv 1 ◽  
In Cells

Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs. However, the reports that exist on their activity against different targets have described mixed success. As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzProdirected against the protease (Pro) coding region and RzRTdirected against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli. Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro. RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzProand RzRTtarget sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT. The RT activity in cells expressing RzRTwas shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any, that could enhance ribozyme activity in vivo.Key words: HIV, hammerhead ribozyme, protease, reverse transcriptase.

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

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