N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by N-myristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30- and 130-fold lower K m values for Nef than Gag, respectively. Values for K cat indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower K m for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef–sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef–sgGFP, whereas depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef–sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence.

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Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

Journal of Virology ◽

10.1128/jvi.78.5.2601-2605.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2601-2605 ◽

Cited By ~ 315

Author(s):

Atze T. Das ◽

Thijn R. Brummelkamp ◽

Ellen M. Westerhout ◽

Monique Vink ◽

Mandy Madiredjo ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Interference ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Antiviral Drug ◽

Target Sequence ◽

Small Interfering Rnas ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

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Human Immunodeficiency Virus Type 1 Escape from RNA Interference

Journal of Virology ◽

10.1128/jvi.77.21.11531-11535.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11531-11535 ◽

Cited By ~ 293

Author(s):

Daniel Boden ◽

Oliver Pusch ◽

Frederick Lee ◽

Lynne Tucker ◽

Bharat Ramratnam

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Interference ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Genome ◽

Antiviral Drug ◽

Selective Inhibition ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

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Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies

Journal of Virology ◽

10.1128/jvi.01594-06 ◽

2007 ◽

Vol 81 (5) ◽

pp. 2485-2496 ◽

Cited By ~ 37

Author(s):

Guerau Fernàndez ◽

Bonaventura Clotet ◽

Miguel Angel Martínez

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fitness Landscape ◽

Catalytic Efficiency ◽

Viral Population ◽

Protease Gene ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected naïve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.

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Potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) by 5-ethyl-6-phenylthiouracil derivatives through their interaction with the HIV-1 reverse transcriptase.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.6.2356 ◽

1991 ◽

Vol 88 (6) ◽

pp. 2356-2360 ◽

Cited By ~ 108

Author(s):

M. Baba ◽

E. De Clercq ◽

H. Tanaka ◽

M. Ubasawa ◽

H. Takashima ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Selective Inhibition ◽

Immunodeficiency Virus ◽

Hiv 1

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Human Immunodeficiency Virus Type 1-Induced Macrophage Gene Expression Includes the p21 Gene, a Target for Viral Regulation

Journal of Virology ◽

10.1128/jvi.79.7.4479-4491.2005 ◽

2005 ◽

Vol 79 (7) ◽

pp. 4479-4491 ◽

Cited By ~ 72

Author(s):

Nancy Vázquez ◽

Teresa Greenwell-Wild ◽

Nancy J. Marinos ◽

William D. Swaim ◽

Salvador Nares ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Replication ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Kinase Inhibitor ◽

Peroxisome Proliferator ◽

Small Interfering Rnas ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT In contrast to CD4+ T cells, human immunodeficiency virus type 1 (HIV-1)-infected macrophages typically resist cell death, support viral replication, and consequently, may facilitate HIV-1 transmission. To elucidate how the virus commandeers the macrophage's intracellular machinery for its benefit, we analyzed HIV-1-infected human macrophages for virus-induced gene transcription by using multiple parameters, including cDNA expression arrays. HIV-1 infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis, and the cell cycle, among which the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) gene was the most prominent. p21 mRNA and protein expression followed a bimodal pattern which was initially evident during the early stages of infection, and maximum levels occurred concomitant with active HIV-1 replication. Mechanistically, viral protein R (Vpr) independently regulates p21 expression, consistent with the reduced viral replication and lack of p21 upregulation by a Vpr-negative virus. Moreover, the treatment of macrophages with p21 antisense oligonucleotides or small interfering RNAs reduced HIV-1 infection. In addition, the synthetic triterpenoid and peroxisome proliferator-activated receptor γ ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which is known to influence p21 expression, suppressed viral replication. These data implicate p21 as a pivotal macrophage facilitator of the viral life cycle. Moreover, regulators of p21, such as CDDO, may provide an interventional approach to modulate HIV-1 replication.

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Inhibition of Human Immunodeficiency Virus Type 1 Replication in Primary Macrophages by Using Tat- or CCR5-Specific Small Interfering RNAs Expressed from a Lentivirus Vector

Journal of Virology ◽

10.1128/jvi.77.22.11964-11972.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 11964-11972 ◽

Cited By ~ 100

Author(s):

Ming-Ta M. Lee ◽

Glen A. Coburn ◽

Myra O. McClure ◽

Bryan R. Cullen

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Vectors ◽

Expression Vectors ◽

Small Interfering Rnas ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs.

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RNA Interference Directed against Viral and Cellular Targets Inhibits Human Immunodeficiency Virus Type 1 Replication

Journal of Virology ◽

10.1128/jvi.76.24.12963-12973.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12963-12973 ◽

Cited By ~ 148

Author(s):

Rama M. Surabhi ◽

Richard B. Gaynor

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

Rna Interference ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Mammalian Cells ◽

Small Interfering Rnas ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-κB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-κB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.

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