Expression of HIV‐1 <i>nef</i> in yeast: The 27 kDa nef protein is myristylated and fractionates with the nucleus

Macreadie, Ian; Ward, Alister C.; Failla, P.; Azad, Ahmed A.; Grgacic, E. V. L.; McPhee, Dale A.

doi:10.1002/yea.320090602

Cited by 16 publications

(18 citation statements)

References 40 publications

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“…Therefore, the observation in the present study that extracellular soluble Nef induced apoptosis in MVECs is consistent with the bystander effect hypothesis (27), which implicates a viral protein or virally stimulated cellular factors released into the medium as mediators of apoptosis (24,72). Since Nef is synthesized early during initial viral infection and it has been shown that Nef produced by recombinant plasmids is secreted into culture medium (31,50,61), it is possible that intracellular expression of Nef in this study resulted in the release of the protein into the culture, which in turn may have caused the bystander effects. Hence, it could also be said that the induction of apoptosis in MVECs by intracellular Nef is also consistent with the bystander effect hypothesis (27).…”

supporting

confidence: 72%

Human Immunodeficiency Virus Type 1 Nef Potently Induces Apoptosis in Primary Human Brain Microvascular Endothelial Cells via the Activation of Caspases

Acheampong

Parveen

Muthoga

et al. 2005

J Virol

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The lentiviral protein Nef plays a major role in the pathogenesis of human immunodeficiency virus type I (HIV-1) infection. Although the exact mechanisms of its actions are not fully understood, Nef has been shown to be essential for the maintenance of high-titer viral replication and disease pathogenesis in in vivo models of simian immunodeficiency virus infection of monkeys. Nef has also been suggested to play a pivotal role in the depletion of T cells by promoting apoptosis in bystander cells. In this context, we investigated the ability of extracellular and endogenously expressed HIV-1 Nef to induce apoptosis in primary human brain microvascular endothelial cells (MVECs). Human brain MVECs were exposed to baculovirus-expressed HIV-1 Nef protein, an HIV-1-based vector expressing Nef, spleen necrosis virus (SNV)-Nef virus (i.e., SNV vector expressing HIV-1 Nef as a transgene), and the HIV-1 strain ADA and its Nef deletion mutant, ADA⌬Nef. We observed that ADA Nef, the HIV-1 vector expressing Nef, and SNV-Nef were able to induce apoptosis in a dose-dependent manner. The mutant virus with a deletion in Nef was able to induce apoptosis in MVECs to modest levels, but the effects were not as pronounced as with the wild-type HIV-1 strain, ADA, the HIV-1-based vector expressing Nef, or SNV-Nef viruses. We also demonstrated that relatively high concentrations of exogenous HIV-1 Nef protein were able to induce apoptosis in MVECs. Gene microarray analyses showed increases in the expression of several specific proapoptotic genes. Western blot analyses revealed that the various caspases involved with Nef-induced apoptosis are processed into cleavage products, which occur only during programmed cell death. The results of this study demonstrate that Nef likely contributes to the neuroinvasion and neuropathogenesis of HIV-1, through its effects on select cellular processes, including various apoptotic cascades.

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supporting

confidence: 72%

Human Immunodeficiency Virus Type 1 Nef Potently Induces Apoptosis in Primary Human Brain Microvascular Endothelial Cells via the Activation of Caspases

Acheampong

Parveen

Muthoga

et al. 2005

J Virol

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“…Detailed immunohistochemical and subcellular fractionation studies have shown that, while Nef is predominantly plasma membrane localized, significant amounts of Nef protein are also found in the cytoplasm and nucleus (27,32,40,41,43). Similarly, MDM-2 localizes to the nucleus and cytoplasm (4).…”

Section: Discussionmentioning

confidence: 99%

“…In infected cells Nef localizes at the plasma membrane and preferentially associates with the cytoskeleton, but it is also found in the cytoplasm, at the nuclear membrane, and in the nucleus (27,32,40,41,43). Three key functions of Nef, which are potentially interrelated, may explain its contribution to disease.…”

mentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 Nef Binds to Tumor Suppressor p53 and Protects Cells against p53-Mediated Apoptosis

Greenway

McPhee

Allen

et al. 2002

J Virol

123

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The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking p53-mediated apoptosis.

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“…The HIV-1 genomic clone pNL4-3 (23) was the source of HIV-1 genes that were amplified by polymerase chain reaction (PCR). The cloning of vpu and nef has been described (24,25). The cloning of vpr is described in Fig.…”

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confidence: 99%

A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects.

Macreadie¹,

Castelli²,

Hewish³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

124

115

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Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cereviswae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two

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Expression of HIV‐1 nef in yeast: The 27 kDa nef protein is myristylated and fractionates with the nucleus

Cited by 16 publications

References 40 publications

Human Immunodeficiency Virus Type 1 Nef Potently Induces Apoptosis in Primary Human Brain Microvascular Endothelial Cells via the Activation of Caspases

Human Immunodeficiency Virus Type 1 Nef Potently Induces Apoptosis in Primary Human Brain Microvascular Endothelial Cells via the Activation of Caspases

Human Immunodeficiency Virus Type 1 Nef Binds to Tumor Suppressor p53 and Protects Cells against p53-Mediated Apoptosis

A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects.

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