Effect of Derivatized Nucleotide Antiviral Drugs on HIV-1 Replication in Cells of T-Lymphoblastoid and Monocyte/Macrophage Lineage in Vitro

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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Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

Journal of Virology ◽

10.1128/jvi.00149-16 ◽

2016 ◽

Vol 90 (15) ◽

pp. 6906-6917 ◽

Cited By ~ 20

Author(s):

Pablo Garcia-Miranda ◽

Jordan T. Becker ◽

Bayleigh E. Benner ◽

Alexander Blume ◽

Nathan M. Sherer ◽

...

Keyword(s):

Thermodynamic Stability ◽

Virus Infectivity ◽

Ribosomal Frameshift ◽

Stem Loop ◽

Assembly Mechanism ◽

Polyprotein Precursor ◽

The Stability ◽

Hiv 1 ◽

In Cells

ABSTRACTHuman immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previousin vitromeasurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication.IMPORTANCEHIV, like many retroviruses, utilizes a −1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

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

mBio ◽

10.1128/mbio.01769-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

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.

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HSF1 inhibition attenuates HIV-1 latency reversal mediated by several candidate LRAs In Vitro and Ex Vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916290117 ◽

2020 ◽

Vol 117 (27) ◽

pp. 15763-15771 ◽

Cited By ~ 1

Author(s):

Andrew Timmons ◽

Emily Fray ◽

Mithra Kumar ◽

Fengting Wu ◽

Weiwei Dai ◽

...

Keyword(s):

High Throughput Screening ◽

Ex Vivo ◽

In Vitro Models ◽

Model Systems ◽

Heat Shock Factor 1 ◽

Primary Cells ◽

Major Barrier ◽

Hiv 1 ◽

In Cells

HIV-1 latency is a major barrier to cure. Identification of small molecules that destabilize latency and allow immune clearance of infected cells could lead to treatment-free remission. In vitro models of HIV-1 latency involving cell lines or primary cells have been developed for characterization of HIV-1 latency and high-throughput screening for latency-reversing agents (LRAs). We have shown that the majority of LRAs identified to date are relatively ineffective in cells from infected individuals despite activity in model systems. We show here that, for diverse LRAs, latency reversal observed in model systems involves a heat shock factor 1 (HSF1)-mediated stress pathway. Small-molecule inhibition of HSF1 attenuated HIV-1 latency reversal by histone deactylase inhibitors, protein kinase C agonists, and proteasome inhibitors without interfering with the known mechanism of action of these LRAs. However, latency reversal by second mitochondria-derived activator of caspase (SMAC) mimetics was not affected by inhibition of HSF1. In cells from infected individuals, inhibition of HSF1 attenuated latency reversal by phorbol ester+ionomycin but not by anti-CD3+anti-CD28. HSF1 promotes elongation of HIV-1 RNA by recruiting P-TEFb to the HIV-1 long terminal repeat (LTR), and we show that inhibition of HSF1 attenuates the formation of elongated HIV-1 transcripts. We demonstrate that in vitro models of latency have higher levels of the P-TEFb subunit cyclin T1 than primary cells, which may explain why many LRAs are functional in model systems but relatively ineffective in primary cells. Together, these studies provide insights into why particular LRA combinations are effective in reversing latency in cells from infected individuals.

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