[Pathogenesis of HIV infections].
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Anti-HIV Agents↗
Biomedical subjects
K Saksela
Publications and source records attributed to K Saksela.
HIV-1 messenger RNA in peripheral blood mononuclear cells as an early marker of risk for progression to AIDS.
OBJECTIVE: To establish human immunodeficiency virus type 1 (HIV-1) messenger RNA (mRNA) expression in peripheral blood mononuclear cells as a marker of risk for progression to the acquired immunodeficiency syndrome (AIDS) in a large cohort of HIV-infected persons followed for a prolonged period. DESIGN: Retrospective testing of cryopreserved, coded specimens. SETTING: Research laboratories at the New York Blood Center and the Rockefeller University. PATIENTS: 150 homosexual men infected with HIV-1 who did not have an AIDS diagnosis at the time of testing. MEASUREMENTS: Multiply spliced and unspliced HIV-1 mRNAs in total peripheral blood mononuclear cell RNA were quantitated using reverse transcriptase-initiated polymerase chain reaction (PCR) and compared with other laboratory data and clinical outcome during the subsequent 8 years. RESULTS: Although HIV-1 mRNA expression generally correlated with immunologic status, it was associated with future disease progression independently of CD4+ cell counts or their rate of decrease at the time of sampling. The association of HIV-1 mRNA with disease progression in persons with CD4+ cell counts higher than the median (> 624 cells/mm3) was particularly noteworthy; further variation in the CD4+ cell counts within this group was not prognostically significant. CONCLUSIONS: The expression of HIV-1 mRNA in peripheral blood mononuclear cells is a strong independent marker for future HIV disease progression, even in persons with normal T-cell subsets.
Acquired Immunodeficiency Syndrome↗
A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.
We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.
Amino Acid Sequence↗
Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4.
Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding.
Amino Acid Sequence↗
HIV-1 RNA in blood and pathogenesis of HIV infection.
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Biomarkers↗
Human immunodeficiency virus type 1 mRNA expression in peripheral blood cells predicts disease progression independently of the numbers of CD4+ lymphocytes.
To address the significance of human immunodeficiency virus (HIV) replication in peripheral blood mononuclear cells (PBMCs), we have used reverse transcriptase-initiated PCR to measure HIV-1 mRNA expression in PBMC specimens collected from a cohort of HIV-infected individuals during a long-term prospective study. We found dramatic differences in HIV mRNA expression among individuals with very similar clinical and laboratory indices, and this variation strongly correlated with the future course of the disease. No evidence of viral replication was detected in PBMCs from asymptomatic individuals who, thereafter, had normal levels of CD4+ cells for at least 5 years. Irrespective of whether the CD4+ cell numbers were normal at the time of sampling, abundant expression of HIV-1 mRNA in PBMCs predicted accelerated disease progression within the next 2 years. Thus, independently of what may be the rate of HIV replication in other viral reservoirs, such as lymphatic tissue, the amount of HIV mRNA in PBMCs appears to reflect the subsequent development of HIV disease. We have also used the reverse transcriptase-initiated PCR assay to demonstrate a transient response to 3'-azido-3'-deoxythymidine treatment. Determination of HIV-1 mRNA expression in the PBMCs of infected individuals could, therefore, have significant clinical utility as a prognostic indicator and as a means to guiding and monitoring antiviral therapies.
CD4-Positive T-Lymphocytes↗
Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses.
To study the basis of cellular latency of human immunodeficiency virus (HIV), we have used a recombinant luciferase-encoding HIV (HXB-Luc) to superinfect nonproductively HIV-1-infected human leukemic cell lines. HXB-Luc contains the Photinus pyralis luciferase gene in place of the nef gene and provides a highly sensitive, simple assay for HIV infection and expression. To circumvent any superinfection block in latently infected cells, we also generated viruses pseudotyped with murine leukemia virus amphotropic envelope (HXB-Luc:ampho). The parental uninfected lines, U937 and A3.01, from which the latently infected cell lines U1 and ACH-2, respectively, were derived could be readily infected with pseudotyped or nonpseudotyped reporter viruses. However, superinfection of U1 cells with either HXB-Luc or HXB-Luc:ampho resulted in only low levels of luciferase activity. Like the endogenous provirus, HXB-Luc provirus could be efficiently activated by phorbol ester treatment of HXB-Luc:ampho-superinfected U1 cells. In contrast, superinfection of ACH-2 cells resulted in active expression of the secondarily introduced virus even in unstimulated cells and luciferase production higher than in the parental cell line A3.01. Thus, the proviral latency in U1 cells appears to result from a defect in the cellular environment (a trans effect), whereas the latency in ACH-2 is specific to the integrated provirus and is probably a cis effect due to the site of integration. These results demonstrate distinct modes of proviral latency in these two cell line models and may have implications in our understanding of the regulation and significance of cellular latency in HIV infection.
Base Sequence↗
High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV-1-infected chimpanzees.
We have examined human immunodeficiency virus type 1 (HIV-1) infection in chimpanzees by analyzing HIV-1 DNA and RNA in lymph nodes and peripheral mononuclear cells (PBMCs). Like certain asymptomatic HIV-infected persons, these chimpanzees had no detectable viral replication in their PBMCs. However, viral replication and a high viral load were observed in the lymphatic tissue. Despite the absence of viral replication in PBMCs, 1/1,000 to 1/10,000 of the PBMCs contained HIV-1 proviral DNA, and HIV transcription could be rapidly induced in these cells in vitro. These results provide direct evidence of cellular latency of HIV in vivo and suggest that HIV infection in chimpanzees may be a useful model for clinical latency of HIV infection in humans.
Animals↗
Negative regulation of immunoglobulin kappa light-chain gene transcription by a short sequence homologous to the murine B1 repetitive element.
B-cell-specific expression of the immunoglobulin kappa light-chain (Ig kappa) gene is in part accomplished by negative regulatory influences. Here we describe a new negatively acting element (termed kappa NE) immediately upstream of the NF-kappa B-binding site in the Ig kappa intronic enhancer. The 27-bp kappa NE sequence is conserved in the corresponding positions in the rabbit and human Ig kappa genes, and the human kappa NE homolog was shown to have a similar negative regulatory activity. Data base searches using the mouse kappa NE sequence revealed a striking homology to murine B1 repetitive sequences. A sequence homologous to kappa NE and B1 was also noted in a previously identified silencer element in the murine T-cell receptor alpha locus. The homologous T-cell receptor alpha locus sequence, but notably not a corresponding 27-bp B1 consensus sequence, showed a negative regulatory potential similar to that of kappa NE. The negative effect of kappa NE by itself was not cell type specific but became so when paired with its 5'-flanking sequence in the Ig kappa enhancer. A short (30-bp) fragment upstream of kappa NE (termed kappa BS) was found to be necessary and sufficient for abolishing the negative effect of kappa NE in B cells. Point mutations in a T-rich motif within the kappa BS sequence allowed the transcriptional repression by kappa NE to be evident in B cells as well as other cells. As suggested by this cell-independent negative activity, proteins binding to the mouse and human kappa NE sequences were identified in all cell types tested.
Animals↗
Amplification and rearrangement of L-myc in human small-cell lung cancer.
DNA amplification of cellular proto-oncogenes is a well-established and common mechanism of oncogene activation in several types of human tumors, including the rapidly fatal small-cell lung cancer (SCLC). Approximately one fourth of primary SCLC tumors contain amplified copies of one of the three myc proto-oncogenes. Occasionally DNA amplification of the myc genes is associated with DNA rearrangements. Specifically, a novel locus named rlf is often involved in intrachromosomal L-myc rearrangements in SCLC. The structurally similar rearrangements are probably due to a highly repetitive region upstream of the L-myc gene, and result in the formation of a chimeric rlf-L-myc fusion protein. The consistent finding of the rlf-L-myc rearrangement in SCLC suggests that it may provide a selective advantage to the cells harboring it.
Carcinoma, Small Cell↗
Activation of protein kinase C increases phosphorylation of the L-myc trans-activator domain at a GSK-3 target site.
The L-myc protein migrates as three distinct differentially phosphorylated bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This phosphorylation can be rapidly increased either by treatment with the protein kinase C (PKC) activator phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by inhibition of serine/threonine protein phosphatases with okadaic acid. In vitro mutagenesis and phosphoamino acid analyses define the N-terminal serine residues 38 and 42 of L-myc as critical targets for the PKC-dependent phosphorylation. These are the exclusive sites of phosphorylation in the N-terminal third of the L-myc protein, and can be phosphorylated in vitro by glycogen synthase kinase 3 beta (GSK-3 beta). A mutant L-myc protein in which these serines have been replaced by alanine residues does not show heterogeneous electrophoretic migration or hyperphosphorylation in response to PKC activation, and is not a substrate for GSK-3 beta in vitro. Similar potential phosphorylation sites are present in c-myc and N-myc in a highly conserved region thought to represent a transcriptional activation domain. We suggest that N-terminal phosphorylation of the L-myc protein is a means of rapid regulation of this oncoprotein, possibly mediated in vivo by the action of GSK-3.
Calcium-Calmodulin-Dependent Protein Kinases↗
A fusion protein formed by L-myc and a novel gene in SCLC.
Oncogenic activation of myc genes in human cancer involves deregulated expression of myc proteins with no major structural alterations. Here two independent small cell lung carcinoma (SCLC) cell lines were found to express similar novel proteins antigenically related to L-myc. cDNAs corresponding to these proteins were cloned and shown to encode chimeric polypeptides with amino-terminal sequences from a novel gene named rlf joined to the L-myc protein. Although the chimeric mRNAs were shown to be identical, they result from distinct DNA rearrangements. The L-myc fusion protein may represent another activation mechanism of the myc proto-oncogenes.
Amino Acid Sequence↗
myc, max, and a novel rlf-L-myc fusion protein in small-cell lung cancer.
The functional properties of Myc proteins are likely to be modulated by interactions with other nuclear proteins. One such protein called Max has already been characterized (1). Through their homologous helix-loop-helix and leucine zipper structures, Myc and Max proteins form heterodimers that bind to specific DNA sequences more efficiently than Myc or Max alone. We have recently identified delta Max, a naturally occurring truncated version of Max, which is also able to dimerize with Myc in the nucleus, but is cytoplasmic in the absence of Myc. These two forms of Max can act either as enhancers or suppressors of cotransformation by c-myc and ras. Oncogenic activation of myc genes in human cancer involves deregulated myc expression. Oncogenes of the myc family are activated in several types of human tumors as a result of gene amplification or chromosomal translocation. We have recently characterized a gene fusion and a chimeric protein product formed by L-myc and part of a novel gene called rlf in small-cell lung cancer (SCLC) cell lines. Although the chimeric mRNAs were shown to be identical, they result from distinct DNA rearrangements. We have also established a physical linkage between normal rlf and L-myc using pulsed field gel electrophoresis. Thus, the rlf-L-myc gene fusions are due to similar but not identical intrachromosomal rearrangements at 1p32. Similar in vivo rearrangements involving rlf and L-myc have been found in at least one primary SCLC tumor. The presence of independent genetic lesions that cause the formation of identical chimeric rlf-L-myc proteins suggests a role for the fusion protein in the development of these SCLC tumors.
Amino Acid Sequence↗
Binding of a nuclear factor to the upstream region of the c-myc gene.
We have studied the binding of proteins to a synthetic DNA fragment corresponding to a 21 base pair sequence in the upstream region of the human c-myc gene. This sequence has previously been reported to bind the c-myc protein, thus representing a putative site for autoregulation of the c-myc gene (Ariga et al., 1989). Our electrophoretic mobility shift assays indicated that this DNA fragment binds to a factor present in nuclear extracts from HeLa cells. However, analysis of extracts from other cells indicated that this binding does not correlate with the presence of the c-myc protein. No binding was observed using c-myc protein translated in vitro. Also, anti-c-myc antibodies could not inhibit binding of this factor. These experiments indicate, that nucleotides -2193 to -2173 from the P1 promoter of the c-myc gene are able to bind a nuclear factor, and that this factor most likely does not include the c-myc protein.
Blotting, Western↗
myc genes and their deregulation in lung cancer.
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Animals↗
Oncogene expression in small-cell lung cancer cell lines and a testicular germ-cell tumor: activation of the N-myc gene and decreased RB mRNA.
N-myc and L-myc proto-oncogenes are expressed in many developing embryonic tissues. In contrast to expression of the closely related c-myc gene, N-myc and L-myc expression is very restricted in adult tissues. We show that small amounts of the L-myc RNA can be detected in normal adult testicular tissue. A high level of N-myc expression from a single-copy N-myc gene was found in a malignant tumor of the testis, histopathologically defined as a seminoma. This tumor also showed a decrease of mRNA from the retinoblastoma gene (RB), which is ubiquitously expressed in all normal tissues. Strikingly similar results on elevated expression of the L-myc and N-myc genes and a lack of RB mRNA have been reported in small-cell lung cancer cell lines, and are confirmed in our study.
Carcinoma, Small Cell↗
Heat shock protects WEHI-164 target cells from the cytolysis by tumor necrosis factors alpha and beta.
Elevated temperatures and a number of other types of stress induce synthesis of a small number of highly conserved proteins, the heat shock proteins, in a wide variety of cells. The structure and regulation of these proteins have been intensively studied but the question of the function of this universal response has remained unanswered. We studied the effect of heat shock on tumor necrosis factor-alpha (TNF-alpha)- and -beta (TNF-beta)-mediated cytolysis of WEHI-164 clone 13 target cells. One hour pretreatment of target cells at 42 degrees C decreased rTNF-alpha-mediated lysis by 65.3%, 50.5% and 44.8% and TNF-beta-mediated lysis by 61.9%, 43.2% and 38.9% at cytokine concentrations of 0.5 ng/ml, 5 ng/ml and 50 ng/ml, respectively, in an 18-h Cr-release assay. The effect was maximal when TNF-alpha was added 1 h after the heat shock and then gradually declined, being almost undetectable after 2 days. This pattern was found to roughly coincide with the kinetics of hsp68, the major heat-induced protein in murine cells. Heat shock treatment had no protective effect when given 1 h after addition of recombinant TNF-alpha. The heat-induced target cell resistance was not associated with decreased binding of recombinant TNF-alpha to its receptor. Inhibition of protein synthesis by cycloheximide diminished this effect by 76% and inhibition of transcription by actinomycin D abolished it completely, suggesting that de novo synthesized heat-induced proteins protect target cells from TNF-mediated lysis in heat shock-treated WEHI cells.
Animals↗
Rapid phosphorylation of the L-myc protein induced by phorbol ester tumor promoters and serum.
We have examined post-translational modification of the L-myc protein using polyclonal and monoclonal antibodies against a peptide well conserved in the predicted amino acid sequences of the c-myc, N-myc and L-myc genes. These antibodies precipitate three polypeptides of Mr 60-66,000 from [35S]methionine or [32P]orthophosphate-labelled human small cell lung cancer cell lines expressing amplified L-myc genes, but not the other myc genes. Treatment of the L-myc immunoprecipitates with alkaline phosphatase prior to electrophoresis converts the three methionine-labelled polypeptides into a single band migrating at Mr 59,000, and efficiently removes radioactivity from the 32P-labelled L-myc protein, suggesting that, in contrast to the c-myc and N-myc proteins, the L-myc polypeptide heterogeneity is due to differential phosphorylation of a common precursor. When the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or serum is added to cultures of U-1690 cells the Mr 66,000 polypeptide is rapidly enriched while the Mr 60,000 form is decreased in the L-myc immunoprecipitates. This effect is correlated with the ability of phorbol ester and diacylglycerol analogues to activate protein kinase C. The TPA-induced phosphorylation of the L-myc protein occurs in a protein synthesis-independent manner as it is not inhibited by cycloheximide or anisomycin. These data indicate that the phosphorylation of the L-myc nuclear oncoprotein is modulated in response to TPA via a rapid signal transduction system involving protein kinase C. This mechanism could play an important role in the response of lung cells to e.g. bombesin-related growth factors.
Antibodies, Monoclonal↗