Nef Expression Research Articles

Interference of HIV-1 Nef in the sphingomyelin transduction pathway activated by tumour necrosis factor-alpha in human glial cells.

The HIV-1 nef gene product, thought to interact with mediators of cell signalling, is overexpressed during the restricted HIV-1 infection of human astrocytes. This infection can be reactivated following exposure to tumour necrosis factor (TNF)-alpha. We examined the possibility that Nef alters the TNF-alpha-induced cell signalling in astroglioma cells through the sphingomyelin pathway. Sphingomyelinase activation by TNF-alpha was analysed in U251MG glial cells constitutively expressing Nef and compared with U251MG cells stably transfected with the expression vector alone. The consequent effect on the cellular proliferative response and induction of nuclear factor NF-kappa B and AP-1 binding activities were examined. A marked enhancement in the levels of ceramide, a product of the sphingomyelin hydrolysis, was observed in U251MG-Nef upon stimulation with TNF-alpha. In contrast, ceramide levels in control cells were barely increased under similar conditions. A concomitant reduction of sphingomyelin level occurred in U251MG-Nef cells. In addition, the reduced survival rate of U251MG cells resulting from TNF-alpha activation was prevented in the presence of Nef. Furthermore, electrophoretic mobility shift assays indicated that nef expression inhibits AP-1 activation without altering the induction of NF-kappa B. These results strongly suggest that nef expression in U251MG cells modulates the sphingomyelinase signalling pathway triggered by TNF-alpha, thus leading to important modifications in the activation and proliferation of glial cells. They also provide new insights to explain the widespread reactive astrogliosis observed in AIDS-associated neuropathological disorders.

Evidence for intact CD28 signaling in T cell hyporesponsiveness induced by the HIV-1 nef gene.

Infection by human immunodeficiency virus (HIV)-1 is associated with quantitative and qualitative T cell alterations that severely impair the host's immune defense system. The molecular basis for this immunosuppression remains unclear. Peripheral blood mononuclear cells (PBMC) isolated from patients show markedly decreased interleukin (IL)-2 secretion but unaffected or even increased T helper (Th)2 cytokine production. T cell functional defects were recently reported to correlate more with T cell receptor (TcR) signaling, whereas signals provided by ligation of co-receptors CD27 and CD28 appeared to be preserved. Among the various mechanisms proposed to be involved in HIV-1-induced T cell dysfunction, we and others have reported that the nef gene product exhibited significant immunosuppressive activity. By using an inducible stably integrated nef gene, we demonstrated that Nef specifically down-regulated IL-2 and interferon (IFN)-gama produced upon TcR triggering. Here, using the same experimental system, we extended our initial observations to additional mitogenic signals, and investigated the co-stimulatory function of CD28. Nef down-regulated IL-2, but not IL-4 produced upon induction by combinations of mitogens that mimicked TcR signals together with CD28 mAb or CD28's natural ligand (CD80 and CD86). However, the co-signals provided by CD28 to up-regulate IL-2 induction were unaffected by Nef, since IL-2 produced by nef-transfected cells was proportionally enhanced to the same extent as that of control cells, either upon stimulation by the CD28 mAb or CD80 and CD86. In addition, phosphatidylinositol-3 kinase recruitment induced upon CD28 triggering was also found to be unaltered by nef expression. Together with the observation that similar levels of the Nef protein were detected in nef-transfected cells and upon infection of PBMC, these data suggest a selective immunosuppression induced by nef in human T cells by altering TcR signaling without detectable impact on CD28 co-receptor function. These data agree with the T cell defects observed in PBMC isolated from HIV-infected individuals.

The HIV Nef Protein Associates with Protein Kinase C Theta

Expression of the human immunodeficiency virus (HIV) Nef protein has been linked to both decreased cell surface expression of CD4 and an impairment of signal transduction. The recently reported association of Nef with an unidentified serine kinase provides a clue as to how Nef might exert its effects. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with thetaPKC. The interaction of Nef and thetaPKC is independent of calcium, enhanced by phospholipid activators of PKC and not affected by a PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to produce the usual translocation of thetaPKC from the cytosol to the particulate fraction; translocation of betaPKC and epsilonPKC was unaffected. Indeed, there appears to be a net loss of thetaPKC in Nef-expressing cells following stimulation. The loss of thetaPKC, which may be a result of inhibition of its binding to RACKs due to Nef binding, could contribute to the various impairments of T cell function associated with HIV infection and Nef expression.

Human Immunodeficiency Virus Type 1 Nef Protein Is Incorporated into Virus Particles and Specifically Cleaved by the Viral Proteinase

The Nef protein of primate immunodeficiency viruses is essential for establishing a highly productive pathogenic infectionin vivo.In tissue culture, Nef is not required for infection but enhances viral infectivity. This effect is most pronounced in unstimulated primary lymphocytes and occurs in the early phase of infection prior to viral gene expression. Since Nef expression does not lead to obvious changes in virus composition, it was of interest to analyze whether Nef is incorporated into virus particles. Here, we show that Nef is specifically immunoprecipitated from radioactively labeled human immunodeficiency virus type 1 (HIV-1)-infected cells and virus particle preparations. Quantitative analysis revealed Nef to be incorporated on the order of 10% of reverse transcriptase incorporation, which corresponds to 5 to 10 molecules of Nef per virion. In infected cells, Nef was detected as a full-length 27-kDa protein. In contrast, approximately 50% of particle-associated Nef corresponded to an 18-kDa species which comigrated with the larger product afterin vitrocleavage of purified HIV-1 Nef by the viral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Nef is cleaved concomitantly with viral structural proteins on maturation of virus particles. This cleavage is likely to be functionally significant because it dissociates the conserved core domain from the N-terminal membrane attachment region. Our results suggest that the profound influence of Nef on establishing infection of unstimulated cells in tissue culture andin vivois mediated by virion-associated Nef which functions in early infection before viral gene expression.

Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.

Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.

The HIV-1 Nef Protein Interferes with Phosphatidylinositol 3-Kinase Activation 1

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

Physical and Functional Interaction of Nef with Lck: HIV-1 Nef-INDUCED T-CELL SIGNALING DEFECTS

The nef gene is unique to the primate lentiviruses and encodes a cytoplasmic membrane-associated protein that affects T-cell signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell signaling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lek tyrosine kinase both in vitro and in vivo. The molecular basis for this interaction was analyzed. We show that cell-derived Nef is precipitated in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely participants in this interaction. The precipitation of Nef by the Lck recombinant proteins was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase nor phospholipase Cgamma SH2 domains coprecipitated Nef from T-cells. Finally, depressed Lck kinase activity resulted from the presence of Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal Lck-mediated signaling events. These results provide a molecular basis for the Nef-induced T-cell signaling defect and its role in AIDS pathogenesis.

Regulation of HIV-1 infection in astrocytes: expression of Nef, TNF-alpha and IL-6 is enhanced in coculture of astrocytes with macrophages.

'Restricted' human immunodeficiency virus type (HIV-1) infection of astrocytes is recognized in vivo in some pediatric and adult AIDS brains and in vitro in a small proportion of transfected primary fetal astrocytes. We investigated the extent of HIV-1JR-FL expression in fetal astrocytes and macrophages cultivated alone or together. Peak HIV-1 p24 antigen titres in supernatant fluids of macrophage cultures were increased with monocyte/macrophages from certain donors and were higher when macrophages were cocultivated with astrocytes. Structural HIV-1 gene (gp 41 and pol) products (protein and mRNA) were observed only in macrophages. Ten days after HIV-1JR-FL infection, astrocytes in a monoculture were stained negative or only weakly positive (1-2+) for Nef, whereas in a coculture up to 100% of astrocytes displayed Nef staining (up to 4+) in the cytoplasm. The streptavidine-biotine-peroxidase technique with certain monoclonal antibodies to Nef (Ovod et al, 1992) was specific for infected astrocytes. The intensity of Nef staining was higher in astrocytes cultivated with monocyte/macrophages from certain donors. In the coculture, tumor necrosis factor-alpha (TNF-alpha) was expressed in the astrocyte cytoplasm earlier after coinfection with HIV-1 and cytomegalovirus (CMV) compared to infection with HIV-1 alone. Interleukin-6 (IL-6) was secreted spontaneously and transiently in uninfected cocultures, but in a prolonged fashion following HIV-1 and HIV-1/CMV infections. The interactions between HIV-1- and CMV-infected macrophages and astrocytes lead to upregulation of TNF-alpha and IL-6 and enhancement of productive HIV-1 infection of macrophages and of 'restricted' HIV-1 infection of astrocytes with implications for the pathogenesis of AIDS dementia.

Specific Th1 cytokine down-regulation associated with primary clinically derived human immunodeficiency virus type 1 Nef gene-induced expression.

HIV-1 infection is associated with a progressive and functional decline in the CD4+ lymphoid Th1 subset. Here, we propose that the HIV nef gene product may function as a specific regulator of Th1 cytokine production. By use of a T cell-specific inducible expression system, we show that upon T cell activation, induced nef expression down-regulated both IL-2 and IFN-gamma production in a dose-dependent manner, whereas IL-4, IL-9, IL-13, IL-8, and TNF-alpha production remained unaffected. In addition to this, independent transfected clones expressing various nef genes, including nef sequences amplified directly from an HIV-1 primary clinical isolate, displayed a similar pattern of cytokine expression. The specific Th1 impairment induced by nef, therefore, seems to be an important and conserved feature of HIV-1 infection and may represent a significant function of this viral gene in AIDS pathogenesis.

Abundant expression of HIV Nef and Rev proteins in brain astrocytes in vivo is associated with dementia

To relate the expression of HIV regulatory proteins and HIV-specific mRNA in the brain cells of infected individuals with clinical neurological disease. Formalin-fixed postmortem brain tissue from 14 HIV-infected adult patients, with previous repeated neurological and neuroradiological examinations, was studied by immunohistochemical and molecular biological methods. Samples from non-infected brains served as controls. Immunohistochemistry with monoclonal antibodies (MAb) was combined with in situ RNA hybridization. Target cells were identified with MAb to glial fibrillary acidic protein (GFAP; astrocytes), CD68 (activated macrophages) and Ricinus communis agglutinin (RCA-1; microglia, endothelial cells). For HIV, a panel of MAb against HIV Nef, Tat, Rev and Env proteins or probes specific for all classes of mRNA (nef), for singly or non-spliced mRNA (env) and for non-spliced mRNA (gag/pol) were used. Nef protein was detected in subcortical or subpial astrocytes in seven out of 14 samples, and in multinucleated giant cells in two cases. Gag/pol or env mRNA-expressing astrocytes were detected in four cases. In four out of five cases studied, HIV Rev, but not Tat, was also expressed in astrocytes. Six out of the seven patients with Nef-positive astrocytes had suffered from moderate to severe dementia. The patient with most rapidly progressing severe dementia showed extensive HIV mRNA expression together with Nef and Rev expression in astrocytes. In adult human brain, astrocytes are infected by HIV and preferentially express HIV Nef and Rev proteins but are also sometimes productively infected. Astrocyte infection is associated with moderate to severe dementia which agrees with recent knowledge on the housekeeping activities of astrocytes and their eventual role in learning and memory.

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines.

Information concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6-9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12-48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.

The HIV-1 regulatory protein Nef has a specific function in viral expression in a murine macrophage cell line.

Expression of reporter genes under the control of the HIV-1 long terminal repeat (LTR) was up-regulated in the murine macrophage cell line RAW264 by cotransfection of a plasmid coding for the viral regulatory protein Nef. To determine if a discrete section of the LTR was exclusively responsive to Nef, a series of promoters was produced by successive 5' deletions from the LTR up to the boundary of the enhancer region. These truncated promoters were as active as the full-length sequence in the RAW264 cells, but elimination of the direct repeats and one of the three Sp1 sites reduced promoter activity to minimal levels. Transcription driven by all constructs was equally susceptible to the trans-activating effect of Nef and could be increased further by the addition of a Tat-expressing plasmid to the cotransfection. Open reading frames of nef from NL4-3, from HXB2, which has a premature stop, and a fully functional hybrid of the two under the control of the SR alpha artificial promoter (SV40 early promoter plus HTLV-I R-U5') were able to transactivate the LTR in RAW264 cells to the same degree as HXB3 nef under the control of the cytomegalovirus (CMV) immediate-early promoter. A frameshift mutation of Nef at the XhoI site at position 8475 did not abrogate trans-activation of the LTR in macrophages. To further define the effective trans-activation region of Nef, internal deletions were made. Changes downstream of the XhoI site at amino acid 35 resulted in little or no reduction in trans-activation, whereas a deletion between the CMV promoter of the expression plasmid and the XhoI site largely abolished activity. Nef trans-activation of the LTR may be restricted to macrophages. Parallel cotransfection experiments in COS-1 simian fibroblast-like cells showed repression of reporter expression by Nef. Results suggested that the section of nef responsible for transactivation of the LTR in macrophages differed slightly from that sufficient for trans-repression in fibroblasts. Translation of the protein from the first translation start site (Met-1) rather than from the second in-frame ATG (Met-20) appears to be necessary for the trans-activating effect of Nef in RAW264 cells. Mutation of the initial ATG to ATA led to loss of trans-activating activity. Expression of Nef also has a cytostatic/cytotoxic effect on RAW264 cells indicated by a reduced rate of establishment of stably transfected clones. The cytostatic effect of Nef was not relieved by internal deletions in the coding sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific Suppression of Human CD4 Surface Expression by Nef from the Pathogenic Simian Immunodeficiency Virus SIVmac239open

HIV-1 Nef down-modulates expression of human CD4, the human immunodeficiency virus (HIV) receptor, at the cell surface. Down-modulation of retrovirus receptors has been shown to be important in the survival of infected cells. To relate this observation to AIDS pathogenesis, we compared the ability of Nef from the SIVmac239open and HIV-1 SF2 isolates to suppress CD4 surface levels. We first obtained the simian immunodeficiency virus (SIV)nef gene by PCR and cloned it into the retroviral vector pLXSN. We then established high titer (1 × 106 CFU/ml) amphotropic retrovirus producer lines (PA317/LSnefSN). Using LSnefSN we obtained populations of CD4+ human and mouse T-cells, human B-cells, and mouse fibroblasts that expressed SIV or HIV Nef. In the two human cell lines, both HIV and SIV Nef expression correlated with a significant decrease in CD4 cell surface levels. However, Nef expression did not alter the cell surface levels of CD3, CD18, and MHC class I. Both Nef proteins also suppressed human CD4 surface expression in mouse fibroblasts. Interestingly, SIV Nef failed to suppress cell surface expression of mouse CD4 under conditions where HIV-1 Nef did. Human CD4 down-modulation is a conserved function of SIV and HIV Nef likely to be important for pathogenesis.

HIV-1 Nef activity in murine T cells. CD4 modulation and positive enhancement.

Immediately after infection of the targeted cell by HIV-1, proviral gene expression is limited to the three regulatory proteins, Tat, Rev, and Nef, with the nef transcript representing nearly 80% of total expression. Additionally, simian immunodeficiency virus Nef has been shown to be essential for high in vivo titer and the development of immunodeficiency. Recent findings demonstrate that the negative effects of Nef expression, as first defined in transformed T cell lines, are not present when Nef is expressed in primary human T cells or in T cells from transgenic mice, in which one sees moderate positive enhancements of HIV replication and the T cell activation process, respectively. We find that Nef expression in an Ag-specific murine T cell hybridoma results in both the down-modulation of CD4, as seen in primary cells and human T cell lines, and a positive enhancement of the TCR response to stimuli. Examination of a CD4- cell demonstrated that the positive enhancement is independent of CD4 expression or modulation. CD4 down-modulation is shown to be caused by a post-Golgi, acid-dependent process, which dramatically decreases the lifespan of the CD4 molecule. The TCR, Thy Ag, and CD45 remained unchanged in their surface expression. These findings suggest that Nef alters the normal routing and residencies of the CD4 molecule and that the positive effect of Nef on T cell activation is independent of this modulation.

Role of HIV-1 Nef Expression in Activation Pathways in CD4+T Cells

The role of the human immunodeficiency virus (HIV-1) Nef protein in T cell activation pathways was investigated using a Jurkat CD4+ cell line stably transfected with a Nef expression vector. Secretion of IL-2 and TNF-alpha, surface expression of IL-2R, and DNA-binding activity of NF-kappa B and AP-1 (Fos/Jun) complex in response to phorbol myristate acetate, TNF-alpha, or immobilized antibodies to CD3 were monitored. These parameters were not modified by Nef expression in Jurkat cells, whereas stimulation with the same stimuli resulted in partial inhibition of LTR activation in Nef+ Jurkat cells. This inhibition was not mediated through Nef phosphorylation on Thr-15 or GTP-binding activity because mutations in critical sites did not alter this inhibition. Analysis of truncated LTRs confirmed that inhibition of LTR activation was not mediated through NF-kappa B-binding activity but through the region containing the negative responding elements (NREs). These results suggest that Nef downmodulates LTR activation without significantly inhibiting the capacity of T cells to respond to immunological activations.

HIV-1 Nef inhibits a common activation pathway in NIH-3T3 cells.

The human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated 27-kDa, cytoplasmic protein. It is attributed to have suppressive effects on LTR-based expression and T cell activation. Additionally, SIV nef has been shown to possess an essential in vivo function in the development of immunodeficiency. To define the biochemical activity of HIV-1 Nef in a signal transduction pathway, we have transduced murine NIH-3T3 cells with a retroviral nef expression system. In nef-expressing cells, but not in controls, the proliferative response to bombesin and platelet-derived growth factor (PDGF) was eliminated. Analysis of an early signal pathway metabolite, inositol 1,4,5-trisphosphate, following bombesin and PDGF treatment to quiscent cells, revealed that both control and nef-transformed cells displayed similar kinetics of signal formation. Normally, inositol 1,4,5-trisphosphate mediates increase in the cytosolic free Ca2+ ([Ca2+]i). Upon stimulation with bombesin or PDGF, control cells displayed a 2-4-fold increase of [Ca2+]i over the basal level, while the [Ca2+]i response in nef-expressing NIH-3T3 cells was lacking or highly diminished. However, the release of [Ca2+]i from the intracellular store of the nef-expressing cells by an endomembrane Ca2+ ATPase inhibitor, thapsigargin, revealed that these cells contained normal Ca2+ stores. These results suggest a specific, definable biochemical activity for the HIV-1 Nef protein in the context of a well characterized cellular activation pathway. Our results thus define, for the first time, a unique function of Nef that is not limited to an alteration of T cell function or of expression of a T cell surface antigen.

Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages

The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells, and many of the colonies that were formed exhibited very slow growth. The frameshift mutant was also active in reducing colony generation. Given the absence of any effect of the frameshift mutation on nef function, its actions on macrophage growth and HIV transcription are discussed in terms of the role of the N-terminal 30 amino acids and of stable secondary structures in the mRNA.

Spontaneous and Inducible Epidermal Hyperplasia in Transgenic Mice Expressing HIV-1 Nef

The pathophysiological consequence of HIV-1 nef gene expression was investigated in transgenic mice carrying a cDNA for Nef linked to either the HIV-1 LTR or the MMTV LTR. In HIV/Nef transgenic mouse lines, nef expression was detected exclusively in the skin and a significant fraction of HIV/Nef transgenic animals (30-75%, depending on the line) spontaneously developed discrete proliferative skin lesions resembling papillomas that were often accompanied by a progressive ulceration of the epidermal cell layer. Nef protein was detected in the basal cell layer of the epidermis and was elevated in the proliferating epidermis. Epidermal cell proliferation could be induced by UV-C irradiation of HIV/Nef transgenic animals but not control mice. An increase in nef expression in the skin accompanied this proliferation. MMTV/Nef mouse lines expressed Nef RNA and protein in organs typically permissive for MMTV LTR-directed transcription but with no obvious pathological consequence.

Expression of HIV-1 nef in yeast: the 27 kDa Nef protein is myristylated and fractionates with the nucleus.

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.