David Baltimore

NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-κB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3′ UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.

Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Double-stranded RNAs ≈21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

Ordered rearrangement of immunoglobulin heavy chain variable region segments.

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ‐line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B‐lymphoid cells as well as normal B‐lymphoid cells. Abelson murine leukemia virus (A‐MuLV) transformed fetal liver cells ‐ the most primitive B‐lymphoid cell analog available for analysis ‐ generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain‐producing tumor lines, representing more mature stages of the B‐cell pathway, and normal B‐lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B‐cell differentiation in which D‐to‐JH rearrangements generally occur first and on both chromosomes followed by VH‐to‐DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain‐producing lines and, especially, in normal B‐lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH‐to‐DJH rearrangement.

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

The structure of a ribonucleoprotein complex formed at the 5′‐end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf‐like structure and interact with both uncleaved 3CD, the viral protease‐polymerase precursor, and a 36 kDa ribosome‐associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem‐loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site‐directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic‐protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5′‐end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

Terminal Deoxynucleotidyl Transferase Activity in Human Leukemic Cells and in Normal Human Thymocytes

Peripheral leukocytes from patients with and without leukemia were assayed for presence of terminal deoxynucleotidyl transferase. Activity of this enzyme was detected in circulating leukemic cells from 11 to 13 patients with acute lymphoblastic leukemia, and in one of four with chronic myelogenous leukemia in blast crisis, but not in leukocytes from patients with other kinds of leukemia or in normal leukocytes. Its presence in a patient with chronic myelogenous leukemia in blast crisis lends biochemical support to the suggestion that some patients with chronic myelogenous leukemia undergo a lymphoblastic rather than a myeloblastic crisis. The thymocyte and leukemic-cell enzyme have the same substrate and primer preference. Normal thymocytes and leukemic cells contain two forms of terminal deoxynucleotidyl transferase that can be separated by phosphocellulose chromatography. The enzyme may provide a means for classifying leukemic cells on a biochemical basis independently of classic morphologic and clinical criteria.

Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Modelling T-cell memory by genetic marking of memory T cells in vivo

Immunological memory is the ability of the immune system to respond with enhanced vigour to pathogens that have been encountered in the past. Following infection or immunization, most effector T cells undergo apoptotic cell death, but a small fraction of these cells, proportional to the early antigen load and initial clonal burst size, persist in the host as a stable pool of memory T cells. The existence of immunological memory has been recognized for over 2,000 years, but our understanding of this phenomenon is limited, primarily because memory lymphocytes cannot be unequivocally identified as they lack specific, permanent markers. Here we have developed a transgenic mouse model system whereby memory T cells and their precursors can be irreversibly marked with a reporter gene and thus can be unambiguously identified. Adoptive transfer of marked CD8+ T cells specific for lymphocytic choriomeningitis virus protected naive recipients following viral challenge, demonstrating that we have marked memory T cells. We also show that cytotoxic effector lymphocytes that develop into memory T cells can be identified in the primary response.

N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl

The two major forms of the c‐abl gene differ from their activated counterpart, the v‐abl oncogene of the Abelson murine leukemia virus by the replacement of their N‐terminal sequences with viral gag sequences. Overexpression of p150c‐abl type IV in a retroviral vector similar to Abelson virus does not transform NIH 3T3 fibroblasts, even though it is expressed and myristoylated at levels comparable to pp160v‐abl. Members of a nested set of deletion mutations of the N‐terminus of c‐abl type IV in this expression system will activate abl to transform murine fibroblasts. The smallest of these deletions, delta XB, efficiently transforms lymphoid cells in vitro and causes leukemia in vivo demonstrating that gag sequences are not necessary for abl‐induced leukemogenesis. The delta XB mutation defines an N‐terminal regulatory domain, which shares a surprising homology with chicken oncogene v‐crk and phospholipase C‐II. Although overexpression of the myristoylated form of c‐abl does not transform cells, it nonetheless has a profound effect on cell growth.

A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene

Complementary DNA cloning of the transcripts of the Drosophila clock gene period reveals three distinct transcripts. These result from unusual splicing pathways, one involving a CG 3′ splice site and one resulting in the use of two different reading frames in one exon, and they predict three separate proteins. Two of the cloned cDNAs can restore clock function to mutant arrhythmic flies.

Terminal Transferase as a Predictor of Initial Responsiveness to Vincristine and Prednisone in Blastic Chronic Myelogenous Leukemia

We undertook a prospective trial to evaluate terminal deoxynucleotidyl transferase activity as a predictor of responsiveness to vincristine and prednisone in 22 Philadelphia-chromosome-positive patients with blastic chronic myelogenous leukemia. Thirteen patients were transferase positive, and nine negative. None of the nine negative patients responded, whereas eight of the 13 positive (P = 0.004) responded with complete clearing of peripheral blood blast cells and a return of normal marrow cellularity with less than 5 per cent blast cells. Among transferase-positive patients under 50 years of age the response rate was 78 per cent. Blast-cell morphology (i.e., lymphoblastic versus myeloblastic) had no significant correlation with either responsiveness or terminal transferase activity. The results of this study suggest that responsiveness to vincristine and prednisone in blastic chronic myelogenous leukemia is confined to patients whose leukemic cells are transferase positive.