Comparative Analyses of Intracellularly Expressed Antisense RNAs as Inhibitors of Human Immunodeficiency Virus Type 1 Replication

ABSTRACT The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3′ long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with theenv antisense RNA, followed by the polcomplementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 × 104 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and envantisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than thetrans-dominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

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Steady-State Pharmacokinetics of Amprenavir Coadministered with Ritonavir in Human Immunodeficiency Virus Type 1-Infected Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.118-123.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 118-123 ◽

Cited By ~ 26

Author(s):

Cecile Goujard ◽

Isabelle Vincent ◽

Jean-Luc Meynard ◽

Nathalie Choudet ◽

Diane Bollens ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Steady State ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Time Curve ◽

Concentration Time ◽

Immunodeficiency Virus ◽

The Mean ◽

Hiv 1

ABSTRACT The protease inhibitor (PI) ritonavir is used as a strong inhibitor of cytochrome P450 3A4, which boosts the activities of coadministered PIs, resulting in augmented plasma PI levels, simplification of the dosage regimen, and better efficacy against resistant viruses. The objectives of the present open-label, multiple-dose study were to determine the steady-state pharmacokinetics of amprenavir administered at 600 mg twice daily (BID) and ritonavir administered at 100 mg BID in human immunodeficiency virus type 1 (HIV-1)-infected adults treated with different antiretroviral combinations including or not including a nonnucleoside reverse transcriptase inhibitor (NNRTI). Nineteen patients completed the study. The steady-state mean minimum plasma amprenavir concentration (C min,ss) was 1.92 μg/ml for patients who received amprenavir and ritonavir without an NNRTI and 1.36 μg/ml for patients who received amprenavir and ritonavir plus efavirenz. For patients who received amprenavir-ritonavir without an NNRTI, the steady-state mean peak plasma amprenavir concentration (C max,ss) was 7.12 μg/ml, the area under the concentration-time curve from 0 to 10 h (AUC0-10) was 32.06 μg · h/ml, and the area under the concentration-time curve over a dosing interval (12 h) at steady-state (AUCss) was 35.74 μg · h/ml. Decreases in the mean values of C min,ss (29%), C max,ss (42%), AUC0-10 (42%), and AUCss (40%) for amprenavir occurred when efavirenz was coadministered with amprenavir-ritonavir. No unexpected side effects were observed. As expected, coadministration of amprenavir with ritonavir resulted in an amprenavir C min,ss markedly higher than those previously reported for the marketed dose of amprenavir. When amprenavir-ritonavir was coadministered with efavirenz, amprenavir-ritonavir maintained a mean amprenavir C min,ss above the mean 50% inhibitory concentration of amprenavir previously determined for both wild-type HIV-1 isolates and HIV-1 strains isolated from PI-experienced patients. These data support the use of low-dose ritonavir to enhance the level of exposure to amprenavir and increase the efficacy of amprenavir.

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Mechanisms by Which the G333D Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Facilitates Dual Resistance to Zidovudine and Lamivudine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00904-07 ◽

2007 ◽

Vol 52 (1) ◽

pp. 157-163 ◽

Cited By ~ 28

Author(s):

Shannon Zelina ◽

Chih-Wei Sheen ◽

Jessica Radzio ◽

John W. Mellors ◽

Nicolas Sluis-Cremer

Keyword(s):

Human Immunodeficiency Virus ◽

Steady State ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Steady State Kinetic ◽

Immunodeficiency Virus ◽

State Kinetic ◽

Hiv 1

ABSTRACT Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.

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Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes

Journal of General Virology ◽

10.1099/0022-1317-83-10-2533 ◽

2002 ◽

Vol 83 (10) ◽

pp. 2533-2542 ◽

Cited By ~ 14

Author(s):

Annette M. G. Dirac ◽

Hendrik Huthoff ◽

Jørgen Kjems ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Base Pair ◽

Sequence Motif ◽

Complementary Sequence ◽

Frequent Event ◽

Immunodeficiency Virus ◽

Hiv 1

Retroviruses are prone to recombination because they package two copies of the RNA genome. Whereas recombination is a frequent event within the human immunodeficiency virus type 1 (HIV-1) and HIV-2 groups, no HIV-1/HIV-2 recombinants have been reported thus far. The possibility of forming HIV-1/HIV-2 RNA heterodimers was studied in vitro. In both viruses, the dimer initiation site (DIS) hairpin is used to form dimers, but these motifs appear too dissimilar to allow RNA heterodimer formation. Multiple mutations were introduced into the HIV-2 DIS element to gradually mimic the HIV-1 hairpin. First, the loop-exposed palindrome of HIV-1 was inserted. This self-complementary sequence motif forms the base pair interactions of the kissing-loop (KL) dimer complex, but such a modification is not sufficient to permit RNA heterodimer formation. Next, the HIV-2 DIS loop size was shortened from 11 to 9 nucleotides, as in the HIV-1 DIS motif. This modification also results in the presentation of the palindromes in the same position within the hairpin loop. The change yielded a modest level of RNA heterodimers, which was not significantly improved by additional sequence changes in the loop and top base pair. No isomerization of the KL dimer to the extended duplex dimer form was observed for the heterodimers. These combined results indicate that recombination between HIV-1 and HIV-2 is severely restricted at the level of RNA dimerization.

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Trafficking through the Rev/RRE Pathway Is Essential for Efficient Inhibition of Human Immunodeficiency Virus Type 1 by an Antisense RNA Derived from the Envelope Gene

Journal of Virology ◽

10.1128/jvi.01520-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 940-952 ◽

Cited By ~ 9

Author(s):

Alex M. Ward ◽

David Rekosh ◽

Marie-Louise Hammarskjold

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Antisense Rna ◽

Envelope Gene ◽

Antisense Inhibition ◽

Rev Response Element ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminal-repeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.

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