Comparative replication capacity of raltegravir-resistant strains and antiviral activity of the new-generation integrase inhibitor dolutegravir in human primary macrophages and lymphocytes

Pollicita, Michela; Surdo, Matteo; Santo, Fabiola Di; Cortese, María Francesca; Fabeni, Lavinia; Fedele, Valentina; Malet, Isabelle; Marcelin, Anne–Geneviève; Cálvez, Vincent; Ceccherini‐Silberstein, Francesca; Perno, Carlo Federico; Svicher, Valentina

doi:10.1093/jac/dku144

Cited by 23 publications

(13 citation statements)

References 52 publications

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“…IC 50 values characterizing RAL, EVG and DTG are in the low-nanomolar range 18 , 24 , 46 , below the concentrations used here in the fluorescence-based assay (0.3–0.6 μM) and below the K d value (sub/low-micromolar) characterizing the binding of DTG to the binary IN-DNA complex. Such a K d value is comparable to values obtained for competitive inhibitors (INBI: IN-binding inhibitors) such as styrylquinolines 47 , 48 , which are much less efficient inhibitors (IC 50 values in the low-micromolar range) than noncompetitive INSTIs.…”

Section: Resultsmentioning

confidence: 71%

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

Thierry¹,

Lebourgeois²,

Simon³

et al. 2017

Sci Rep

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FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mg2+ chelation, allowing to directly probe its Mg2+-dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3′-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravir. The dolutegravir-binding properties derived from fluorescence-based binding assays and drug susceptibilities in terms of catalytic activity, are well correlated. Indeed, dolutegravir-binding to wild-type and F190Y integrases are comparable while strongly compromised with G187R and S217K. Accordingly, the two latter mutants are highly resistant to dolutegravir while F190Y shows only moderate or no resistance. Intrinsic fluorescence properties of dolutegravir are thus particularly suitable for a thorough characterization of both DNA-binding properties of integrase and resistance mutations.

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Section: Resultsmentioning

confidence: 71%

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

Thierry¹,

Lebourgeois²,

Simon³

et al. 2017

Sci Rep

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“…To confirm and extend this observation, we infected primary peripheral blood mononuclear cells (PBMCs) and various human cell lines with an NL4-3-based indicator virus in which the nef gene has been replaced with the Nano luciferase (NLuc) indicator gene (NL-NLuc). Cells were infected with WT HIV-1, with an IN mutant (D64V) that lacks integrase function, or with WT HIV-1 in the presence of 20 µM raltegravir (RAL), which blocks IN function (20,21). Levels of NLuc expression were quantified and normalized to WT HIV-1, which was set at 100%.…”

Section: Resultsmentioning

confidence: 99%

Robust HIV-1 replication in the absence of integrase function

Irwan

Karnowski

Bogerd

et al. 2020

Preprint

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Integration of the proviral DNA intermediate into the host cell genome represents an essential step in the retroviral life cycle. While the reason(s) for this requirement remains unclear, it is known that unintegrated proviral DNA is epigenetically silenced. Here, we demonstrate that HIV-1 mutants lacking functional integrase can mount a robust, spreading infection in cells expressing the Tax transcription factor encoded by human T-cell leukemia virus 1. In these cells, HIV-1 forms episomal DNA circles, analogous to Hepatitis B virus covalently closed circular DNAs (cccDNAs), that are transcriptionally active and fully capable of supporting viral replication. This rescue correlates with the loss of inhibitory epigenetic marks, and the acquisition of activating marks, on histones bound to unintegrated HIV-1 DNA. Thus retroviral DNA integration may have evolved, at least in part, as a mechanism to avoid the epigenetic silencing of extrachromosomal viral DNA by host innate antiviral factors. SignificanceWhile retroviral DNA is synthesized normally after infection by integrase-deficient viruses, the resultant episomal DNA is then epigenetically silenced. Here, we show that expression of the Tax transcription factor encoded by a second human retrovirus, HTLV-1, prevents the epigenetic silencing of unintegrated HIV-1 DNA and instead induces the addition of activating epigenetic marks, and the recruitment of NF-kB/Rel proteins, to the HIV-1 LTR promoter. Moreover, in the presence of Tax, the HIV-1 DNA circles that form in the absence of integrase function are not only efficiently transcribed but also support a spreading, pathogenic IN-HIV-1 infection. Thus, retroviruses have the potential to replicate without integration, as is indeed seen with HBV.

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“…First, we reasoned that the combination of sustained peak plasma viremia (Figure 1) and low baseline CD4+ T-cell counts (Figure 3) could have facilitated infection of peripheral blood CD4+ myeloid cells, which could serve as drug sanctuaries [25–27]. No significant differences in average monocyte counts were found in any animal during the course of study except for an elevation in Group III animal A11202 between 6 and 12 weeks post-SHIV challenge (Supplementary Figure 2A–C).…”

Section: Resultsmentioning

confidence: 99%

Lack of viral control and development of combination antiretroviral therapy escape mutations in macaques after bone marrow transplantation

Peterson

Haworth

Polacino³

et al. 2015

Aids

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Objective We have previously demonstrated robust control of simian/human immunodeficiency virus (SHIV1157-ipd3N4) viremia following administration of combination antiretroviral therapy (cART) in pigtailed macaques. Here, we sought to determine the safety of hematopoietic stem cell transplantation (HSCT) in cART-suppressed and unsuppressed animals. Design We compared disease progression in animals challenged with SHIV 100 days post-transplant (PT), to controls that underwent transplant following SHIV challenge and stable, cART-dependent viral suppression. Methods SHIV viral load, combination antiretroviral therapy (cART) levels, and anti-SHIV antibodies were measured longitudinally from plasma/serum from each animal. Flow cytometry was used to assess T-cell subset frequencies in peripheral blood and gastrointestinal tract (GI). Deep sequencing was used to identify cART resistance mutations. Results In control animals, virus challenge induced transient peak viremia, viral set point, and durable suppression by cART. Subsequent HSCT was not associated with adverse events in these animals. PT animals were challenged during acute recovery following HSCT, and displayed sustained peak viremia and cART resistance. Although PT animals had comparable plasma levels of antiretroviral drugs and showed no evidence of enhanced infection of myeloid subsets in the periphery, they exhibited a drastic reduction in virus-specific antibody production and decreased T-cell counts. Conclusions These results suggest that virus challenge prior to complete transplant recovery impairs viral control and may promote drug resistance. These findings may also have implications for scheduled treatment interruption (STI) studies in patients on cART during post-HSCT recovery: premature STI could similarly result in lack of viral control and cART resistance.

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Comparative replication capacity of raltegravir-resistant strains and antiviral activity of the new-generation integrase inhibitor dolutegravir in human primary macrophages and lymphocytes

Cited by 23 publications

References 52 publications

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

Robust HIV-1 replication in the absence of integrase function

Lack of viral control and development of combination antiretroviral therapy escape mutations in macaques after bone marrow transplantation

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