scholarly journals Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

2009 ◽  
Vol 54 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Olivier Delelis ◽  
Sylvain Thierry ◽  
Frédéric Subra ◽  
Françoise Simon ◽  
Isabelle Malet ◽  
...  
Keyword(s):  
Viral Genome ◽  
Effective Concentration ◽  
Coding Region ◽  
Delayed Emergence ◽  
Infected Cells ◽  
High Level ◽  
Emergence Of Resistance ◽  
Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

scholarly journals HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Teslin S. Sandstrom ◽  
Nischal Ranganath ◽  
Stephanie C. Burke Schinkel ◽  
Syim Salahuddin ◽  
Oussama Meziane ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Oncolytic Viruses ◽  
Type I Interferons ◽  
Viral Reservoir ◽  
Type I ◽  
Antiviral Signaling ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus—initially developed as a cancer therapy—is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

scholarly journals How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill?

2005 ◽  
Vol 79 (21) ◽  
pp. 13579-13586 ◽  
Author(s):  
W. David Wick ◽  
Otto O. Yang ◽  
Lawrence Corey ◽  
Steven G. Self
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.

scholarly journals In VitroCharacterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile

2011 ◽  
Vol 55 (4) ◽  
pp. 1366-1376 ◽  
Author(s):  
Christian Callebaut ◽  
Kirsten Stray ◽  
Luong Tsai ◽  
Matt Williams ◽  
Zheng-Yu Yang ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Cell Types ◽  
Human Serum Protein ◽  
Treatment Naïve ◽  
Low Cytotoxicity ◽  
T Cell Lines ◽  
High Level ◽  
Hiv 1

ABSTRACTGS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to theparaposition of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki= 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50= 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs,in vitrohepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boostingin vivo. In summary, results from this broadin vitropharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

scholarly journals A Therapeutic Strategy to Combat HIV-1 Latently Infected Cells With a Combination of Latency-Reversing Agents Containing DAG-Lactone PKC Activators

2021 ◽  
Vol 12 ◽  
Author(s):  
Kouki Matsuda ◽  
Takuya Kobayakawa ◽  
Ryusho Kariya ◽  
Kiyoto Tsuchiya ◽  
Shoraku Ryu ◽  
...  
Keyword(s):  
Whole Body ◽  
Therapeutic Effects ◽  
Combined Antiretroviral Therapy ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1 ◽  
Reversing Agents

Advances in antiviral therapy have dramatically improved the therapeutic effects on HIV type 1 (HIV-1) infection. However, even with potent combined antiretroviral therapy, HIV-1 latently infected cells cannot be fully eradicated. Latency-reversing agents (LRAs) are considered a potential tool for eliminating such cells; however, recent in vitro and in vivo studies have raised serious concerns regarding the efficacy and safety of the “shock and kill” strategy using LRAs. In the present study, we examined the activity and safety of a panel of protein kinase C (PKC) activators with a diacylglycerol (DAG)-lactone structure that mimics DAG, an endogenous ligand for PKC isozymes. YSE028, a DAG-lactone derivative, reversed HIV-1 latency in vitro when tested using HIV-1 latently infected cells (e.g., ACH2 and J-Lat cells) and primary cells from HIV-1-infected individuals. The activity of YSE028 in reversing HIV-1 latency was synergistically enhanced when combined with JQ1, a bromodomain and extra-terminal inhibitor LRA. DAG-lactone PKC activators also induced caspase-mediated apoptosis, specifically in HIV-1 latently infected cells. In addition, these DAG-lactone PKC activators showed minimal toxicity in vitro and in vivo. These data suggest that DAG-lactone PKC activators may serve as potential candidates for combination therapy against HIV-1 latently infected cells, especially when combined with other LRAs with a different mechanism, to minimize side effects and achieve maximum efficacy in various reservoir cells of the whole body.

scholarly journals Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Hongping Jin ◽  
Yifan Sun ◽  
Dongsheng Li ◽  
Min-Hsuan Lin ◽  
Mary Lor ◽  
...  
Keyword(s):  
Mutant Form ◽  
Mrna Levels ◽  
Functional Cure ◽  
Fold Reduction ◽  
Infected Cells ◽  
Hiv 1 ◽  
Transcription And Replication ◽  
In Cells

ABSTRACT Nullbasic is a mutant form of the HIV-1 transcriptional activator protein (Tat) that strongly inhibits HIV-1 transcription and replication in lymphocytes in vitro. To investigate Nullbasic inhibition in vivo, we employed an NSG mouse model where animals were engrafted with primary human CD4+ cells expressing a Nullbasic-ZsGreen1 (NB-ZSG) fusion protein or ZSG. NB-ZSG and ZSG were delivered by using a retroviral vector where CD4+ cells were transduced either prior to (preinfection) or following (postinfection) HIV-1 infection. The transduced cells were analyzed in vitro up to 10 days postinfection (dpi) and in vivo up to 39 dpi. Compared to ZSG, NB-ZSG strongly inhibited HIV-1 replication both in vitro and in vivo using preinfection treatment. In vitro, HIV-1 mRNA levels in cells were reduced by up to 60-fold. In vivo, HIV-1 RNA was undetectable in plasma samples during the course of the experiment, and HIV-1 mRNA levels in resident CD4+ cells in organ tissue were reduced up to 2,800-fold. Postinfection treatment of HIV-1-infected cells with NB-ZSG attenuated HIV-1 infection for up to 14 days. In vitro, a 25-fold reduction of viral mRNA in cells was observed but diminished to a <2-fold reduction by 10 dpi. In vivo, HIV-1 RNA was undetectable in plasma of NB-ZSG mice at 14 dpi but afterwards was not significantly different between NB-ZSG mice and control mice. However, we observed higher levels of CD4+ cells in NB-ZSG mice than in control mice, suggesting that NB-ZSG imparted a survival advantage to HIV-1-infected animals. IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces viral loads below detectable levels in patients. However, therapy interruption leads to viral rebound due to latently infected cells, which serve as a source of continued viral infection. Interest in strategies leading to a functional cure for HIV-1 infection by long-term or permanent viral suppression is growing. Here, we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, inhibits HIV-1 transcription in infected CD4+ cells in vivo. Analysis shows that stable expression of Nullbasic in CD4+ cells could lead to durable anti-HIV-1 activity. Nullbasic, as a gene therapy candidate, could be a part of a functional-cure strategy to suppress HIV-1 transcription and replication.

scholarly journals A Critical Review of the Evidence Concerning the HIV Latency Reversing Effect of Disulfiram, the Possible Explanations for Its Inability to Reduce the Size of the Latent Reservoir In Vivo, and the Caveats Associated with Its Use in Practice

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Harry D. J. Knights
Keyword(s):  
Immune Deficiency Syndrome ◽  
Deficiency Syndrome ◽  
Latent Reservoir ◽  
Major Barrier ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1

Combination antiretroviral therapy (cART) effectively suppresses the replication of human immunodeficiency virus type 1 (HIV-1), improves immune function, and decreases the morbidity of acquired immune deficiency syndrome (AIDS). However, it is unable to eradicate the virus because it does not eliminate latently infected cells. The latent reservoir poses the major barrier to an HIV-1 cure. The “shock and kill” strategy aims to reactivate the virus and destroy latently infected cells. Many latency reversing agents (LRAs) reactivate HIV in vitro, but the absence of damaging side-effects and efficacy in vivo make disulfiram particularly promising. However, in clinical trials to date, disulfiram treatment has not resulted in a reduction in the size of the latent reservoir. In this article I will therefore discuss the evidence for the latency reversing effect of disulfiram, the possible explanations for its inability to reduce the size of the latent reservoir in vivo, and the caveats associated with its use in practice. These considerations will help to inform judgements about the prospect of an HIV cure from disulfiram based treatments.

scholarly journals Direct proof that the primary site of action of cytochalasin on cell motility processes is actin.

1992 ◽  
Vol 116 (4) ◽  
pp. 933-941 ◽  
Author(s):  
H Ohmori ◽  
S Toyama ◽  
S Toyama
Keyword(s):  
Cell Motility ◽  
Direct Proof ◽  
Primary Site ◽  
Coding Region ◽  
Beta Actin ◽  
Site Of Action ◽  
Altered Form ◽  
High Level

We have previously described the isolation of a mutant KB cell (Cyt 1 mutant) resistant to the cytotoxic effect of cytochalasin B (CB). The Cyt 1 mutant carries an altered form of beta-actin (beta'-actin) and lacks normal beta-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). Increased resistance of the Cyt 1 mutant to CB in vivo is reflected in altered properties of beta'-actin in vitro (Toyama, S., and S. Toyama. 1988. J. Cell Biol. 107:1499-1504). Here, we show that the mutation in beta-actin is solely responsible for the cytochalasin-resistant phenotype of the Cyt mutant. We have isolated a cDNA clone encoding beta'-actin from Cyt 1 cells. Sequence analysis reveals two mutations in the coding region that substitute two amino acid residues (Val139----Met and Ala295----Asp). Expression of the beta'-actin cDNA confers cytochalasin resistance upon transformed cytochalasin-sensitive KB cells. Levels of resistance to CB in the transformed cell clones correlate well with amounts of beta'-actin polypeptide. Both of the two mutations in beta'-actin are necessary for the high level expression of cytochalasin resistance. Overall, we conclude that the primary site of action of cytochalasin on cell motility processes in vivo is actin.

scholarly journals Deletion of the Autographa californica nucleopolyhedrovirus chitinase KDEL motif and in vitro and in vivo analysis of the modified virus

2004 ◽  
Vol 85 (4) ◽  
pp. 821-831 ◽  
Author(s):  
Giles P. Saville ◽  
Alexandra L. Patmanidi ◽  
Robert D. Possee ◽  
Linda A. King
Keyword(s):  
Trichoplusia Ni ◽  
Lethal Dose ◽  
Gene Promoter ◽  
Autographa Californica ◽  
Coding Region ◽  
Insect Larvae ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Infected Cells

Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.

scholarly journals Targeting and Understanding HIV Latency: The CRISPR System against the Provirus

Pathogens ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1257
Author(s):  
Gloria Magro ◽  
Arianna Calistri ◽  
Cristina Parolin
Keyword(s):  
Viral Latency ◽  
In Vivo Studies ◽  
Latently Infected ◽  
Infected Cells ◽  
Specific Factors ◽  
The One ◽  
Cellular Dna ◽  
Hiv 1

The presence of latently infected cells and reservoirs in HIV-1 infected patients constitutes a significant obstacle to achieve a definitive cure. Despite the efforts dedicated to solve these issues, the mechanisms underlying viral latency are still under study. Thus, on the one hand, new strategies are needed to elucidate which factors are involved in latency establishment and maintenance. On the other hand, innovative therapeutic approaches aimed at eradicating HIV infection are explored. In this context, advances of the versatile CRISPR-Cas gene editing technology are extremely promising, by providing, among other advantages, the possibility to target the HIV-1 genome once integrated into cellular DNA (provirus) and/or host-specific genes involved in virus infection/latency. This system, up to now, has been employed with success in numerous in vitro and in vivo studies, highlighting its increasing significance in the field. In this review, we focus on the progresses made in the use of different CRISPR-Cas strategies to target the HIV-1 provirus, and we then discuss recent advancements in the use of CRISPR screens to elucidate the role of host-specific factors in viral latency.

scholarly journals Human MAIT cells respond to and suppress HIV-1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Chansavath Phetsouphanh ◽  
Prabhjeet Phalora ◽  
Carl-Philipp Hackstein ◽  
John Thornhill ◽  
Mee Ling Munier ◽  
...  
Keyword(s):  
Protective Role ◽  
Target Cells ◽  
Restriction Factors ◽  
Innate And Adaptive Immunity ◽  
Mait Cells ◽  
Infected Cells ◽  
Hiv 1 ◽  
Stimulation Of

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1 infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to up-regulate granzyme B, IFNg and HIV-1 restriction factors CCL3, 4 and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

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