Chimeric Human Immunodeficiency Virus Type 1 Virions That Contain the Simian Immunodeficiency Virus
            <i>nef</i>
            Gene Are Cyclosporin A Resistant

Khan, Mahfuz; Jin, Lingling; Miles, Lesa; Bond, Vincent C.; Powell, Michael D.

doi:10.1128/jvi.79.5.3211-3216.2005

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…This early capture may govern the directed movement of vRNA along the cytoskeleton, to the translation apparatus, to sites of viral assembly and finally, into assembling viral particles. A number of host gene products, such as hnRNP A1, PSF/nsr54 and APOBEC3G associate with vRNA (Beriault et al, 2004; Khan et al, 2005). Furthermore, a limited set of host trans -acting proteins mediates trafficking by binding to specific cis -acting sequence elements in vRNA (Mouland et al, 2001; Levesque et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

Milev¹,

Ravichandran²,

Khan³

et al. 2012

Front. Microbio.

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The human immunodeficiency virus type 1 (HIV-1) unspliced, 9 kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as >200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced vRNA in HIV-1-producing cells.

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Section: Introductionmentioning

confidence: 99%

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

Milev¹,

Ravichandran²,

Khan³

et al. 2012

Front. Microbio.

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“…63,64 HEK293 or 293FT cells were transfected with Nef or a Nef-GFP expression plasmid using the Effectene transfection kit (Qiagen) according to the manufacturer's instructions. Briefly, cells were plated in 100-mm dishes and transfected at 90% confluency.…”

Section: Plasmid Constructs and Transfectionsmentioning

confidence: 99%

HIV Type 1 Nef Is Released from Infected Cells in CD45⁺Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

Raymond

Campbell-Sims²,

Khan³

et al. 2011

AIDS Research and Human Retroviruses

148

157

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HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV MN . Interestingly, plasma mvNef levels in HIV þ patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIVinfected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.

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Functional characterization of HIV-1 Nef mutants in the context of viral infection

Moris²,

et al. 2006

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Nef is an important pathogenesis factor of HIV-1 with a multitude of effector functions. We have designed a broad panel of isogenic viruses encoding defined mutants of HIV-1(SF2) Nef and analyzed their biological activity in the context of productive HIV-1 infection. Analysis of subcellular localization, virion incorporation, downregulation of cell surface CD4 and MHC-I, enhancement of virion infectivity and facilitation of HIV replication in primary human T lymphocytes mostly confirmed the mapping of Nef determinants previously reported upon isolated expression of Nef. However, reduced activity in downregulation of CD4, infectivity enhancement and virion incorporation of a Nef variant (Delta12-39) lacking an amphipatic helix required for binding of a cellular kinase complex and the association of Nef with MHC-I/AP-1 suggested a novel role of this N-terminal motif. The SH3 binding motif of Nef was partially required for infectivity enhancement and replication but not for receptor downmodulation. In contrast to previous results obtained using other Nef alleles, non-myristoylated SF2-Nef was only partly defective when expressed during HIV infection and was present in HIV-1 particles. Importantly, incorporation of Nef into HIV-1 virions was not required for any of the tested Nef activities. Altogether, this study provides a broad characterization and mapping of multiple Nef activities in HIV-infected cells. The results emphasize that multiple activities govern Nef's effects on HIV replication and argue against a role of virion incorporation for Nef's activity as pathogenicity factor.

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Chimeric Human Immunodeficiency Virus Type 1 Virions That Contain the Simian Immunodeficiency Virus nef Gene Are Cyclosporin A Resistant

Cited by 3 publications

References 36 publications

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

HIV Type 1 Nef Is Released from Infected Cells in CD45⁺Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

Functional characterization of HIV-1 Nef mutants in the context of viral infection

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Chimeric Human Immunodeficiency Virus Type 1 Virions That Contain the Simian Immunodeficiency Virus nef Gene Are Cyclosporin A Resistant

Cited by 3 publications

References 36 publications

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

HIV Type 1 Nef Is Released from Infected Cells in CD45+Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

Functional characterization of HIV-1 Nef mutants in the context of viral infection

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HIV Type 1 Nef Is Released from Infected Cells in CD45⁺Microvesicles and Is Present in the Plasma of HIV-Infected Individuals