Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product

Kaminchik, J; Bashan, Nava; Pinchasi, Dalia; Amit, Boaz; Sarver, Nava; Johnston, Mark; Fischer, Michael; Yavin, Ziva; Gorecki, Marian; Panet, Amos

doi:10.1128/jvi.64.7.3447-3454.1990

Cited by 72 publications

(29 citation statements)

References 26 publications

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“…It overlaps the 3Ј long terminal repeat (LTR) and in some virus species also the 3Ј-end of env (Harris, 1996;Michael et al, 1995). It is transcribed early in infection from multiply spliced RNAs (Kaminchik et al, 1990), and depending on the virus strain, nef encodes a myristoylated protein of 25±32 kDa that is located on the cytoplasmic side of the cell membrane (Guy et al, 1987;Franchini et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

Macaques Infected with Cloned Simian Immunodeficiency Virus Show RecurringnefGene Alterations

et al. 1998

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We compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2-8) or late (weeks 21-137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located approximately 40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrent in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MA nef) nef on the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnef contained a large proportion of cells carrying provirus with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were "corrected" in vivo.

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Section: Introductionmentioning

confidence: 99%

Macaques Infected with Cloned Simian Immunodeficiency Virus Show RecurringnefGene Alterations

et al. 1998

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“…Nef has been shown to downregulate the IL-2 mRNA (Luria et al, 1991) and the surface expression of the HIV-1 receptor, CD4 (Guy et al, 1990;Garcia and Miller, 1991;Gama Sosa et al, 1991), however, the mechanism of this function is not known. In addition Escherichia coli-derived Nef has been reported to have G protein activities that include GTP binding, GTPase and autophosphorylation (Guy et al, 1987(Guy et al, , 1990) but a lack of some or all of these activities has been reported by others (Kaminchik et al, 1990;Backer et al, 1991;Nebreda et al, 1991;Wolber et al, 1992). In insect cells, the only eukaryotic cells previously used for the production and purification of Nef, these activities were also found to be lacking (Matsuura et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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

Macreadie¹,

Ward²,

Failla³

et al. 1993

Yeast

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The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M(r) 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.

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“…In fact, Kim et al (11) observed that infection of activated CD4 § T lymphocytes with a Nef § viral strain resulted in moderately enhanced viral replication as compared with a nefmutant. Additionally, the mechanistically intriguing observation that Nef both binds GTP and exhibits GTPase activity (12) was subsequently refuted by three different laboratories employing purified Nef protein isolated from both bacterial and insect sources (13)(14)(15). Perhaps the only property of Nef that is not in dispute is its inhibitory effect on cell surface CD4 expression.…”

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confidence: 99%

The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.

Miller

Warmerdam

Gaston

et al. 1994

The Journal of Experimental Medicine

498

385

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SummaryConsiderable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-I nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef + HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nefmutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef + and Nef-viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef-viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact neffor subsequent viral growth. We now provide evidence that the Nef § HIV is more infectious than Nef-HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five-to sixfold more infected cells if Nef + viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef + virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef + and Nef-viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef + infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMCderived Nef + HIV. In summary, these findings demonstrate a consistent positive role for the HIV-1 nef gene in promoting viral infection and replication, and suggest that the basis for this phenotype is the increased infectivity of HIV produced from cells expressing nef. These data suggest that HIV-1 Nef, as previously shown for SIV Nef, may play an important role in establishing a fulminant form of viral infection in vivo.

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Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product

Cited by 72 publications

References 26 publications

Macaques Infected with Cloned Simian Immunodeficiency Virus Show RecurringnefGene Alterations

Macaques Infected with Cloned Simian Immunodeficiency Virus Show RecurringnefGene Alterations

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

The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.

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