Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3' (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.     

Alternate strand DNA triple helix-mediated inhibition of HIV-1 U5 long terminal repeat integration in vitro

Integration of the human immunodeficiency virus (HIV) DNA into the host genome is an obligatory process in the replicative life cycle of the virus. This event is mediated in vitro by integrase, a viral protein which binds to specific sequences located on both extremities of the DNA long terminal repeats (LTRs). These sites are highly conserved in all HIV genomes and thus provide potential targets for the selective inhibition of integration. The integrase-binding site located on the HIV-1 U5 LTR end contains two adjacent purine tracts on opposite strands, 5' . . . GGAAAATCTCT-3'/3'-CCTTTTAGAGA . . . 5', in parallel orientations. A single strand oligonucleotide 5'-GGTTTTTGTGT-3' was designed to associate with these tracts via its ability to form a continuous alternate strand DNA triplex. Under neutral pH and physiological temperature, the oligonucleotide, tagged with an intercalator chromophore oxazolopyridocarbazole, formed a stable triplex with the target DNA. The occurrence of this unusual triplex was demonstrated by both DNase I footprinting and electron microscopy. The triplex inhibits the two steps of the integrase-mediated reactions, namely, the endonucleolytic cleavage of the dinucleotide 5'-GT-3' from the 3' end of the integration substrate and the integration of the substrate into the heterologous target DNA. The midpoints for both inhibition reactions were observed at oligonucleotide concentrations of 50-100 nM. We believe that these results open new possibilities for the specific targeting of viral DNA LTR ends with the view of inhibiting integration under physiological conditions.     

Isolation of new oncogenic forms of the murine c-fms gene

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor, which plays a key role in the proliferation and differentiation of cells of the myelomonocytic lineage. In order to study the effects of overexpression of the macrophage colony-stimulating factor receptor in hematopoietic cells, a Harvey sarcoma virus-derived retroviral vector containing the murine c-fms cDNA was pseudotyped with Friend murine leukemia virus and inoculated into newborn DBA/2 mice. This viral complex induced monoclonal or oligoclonal leukemias with a shorter latency than that for Friend murine leukemia virus alone. Unexpectedly, 60% of the integrated fms proviruses had deletions at the 5' end of the c-fms gene. Sequence analysis of seven mutant proviruses indicated that the deletions always included the c-fms ligand binding domain and either occurred within the c-fms sequences, leaving the fms open reading frame unchanged, or joined VL30 sequences located at the 5' end of the parental retroviral vector to internal c-fms sequences, resulting in truncated fms proteins devoid of the canonical signal peptide. In contrast to all tyrosine kinase receptors transduced in retroviruses, no helper gag- or env-derived sequences were fused to the rearranged fms sequences. Viral supernatants isolated from hematopoietic tumors with viruses with deletions were able to transform NIH 3T3 cells as efficiently as parental fms virus, indicating that deletions resulted in constitutive activation of the c-fms gene. These oncogenic variants differ from those transduced in the Suzan McDonough strain of feline sarcoma viruses (L. Donner, L. A. Fedele, C. F. Garon, S. J. Anderson, and C. J. Sherr, J. Virol. 41:489-500, 1982). The high rate of c-fms rearrangement and its relevance in the occurrence of hematopoietic tumors are discussed.     

Electron microscopic evidence for splicing of Moloney murine leukemia virus RNAs

Poly (A) containing RNA extracted from Moloney murine leukemia virus infected mouse cells was hybridized with long single-stranded complementary DNA, prepared in detergent disrupted virions. Visualization of the hybrids in the electron microscope revealed among the structures, circles and circles with tails. Measurements performed on the circular molecules revealed two major species with circumferences corresponding to 3 and 8.2 kilobases. The latter structures had identical size to circles obtained after annealing of cDNA with the viral genome, 35S RNA. Circularization of a small viral RNA (3 kb) from infected cells in the RNA-cDNA hybrids is a direct evidence that like the 35S RNA it shares similar nucleotide sequences at both the 5' and 3' ends. The presence of 5' end sequences common to the two RNA species indicates the existence of a spliced viral RNA. Furthermore, based on the circularization of viral RNA in the hybrids, we suggest a new way to quantitate and determine the lengths of spliced RNA in retrovirus infected cells.     

Heterogeneous nuclear ribonucleoprotein I (hnRNP-I/PTB) selectively binds the conserved 3' terminus of hepatitis C viral RNA

Hepatitis C virus (HCV) is a positive-strand RNA virus whose genome is replicated by a direct RNA-to-RNA mechanism. Initiation of negative-strand RNA synthesis is believed to proceed from the 3' end of the genomic RNA. The high conservation of the 3' terminus suggests that this region directs the assembly of proteins required for the initiation of RNA replication. We sought to determine whether host proteins bind specifically to this RNA structure. We observed specific binding of cellular proteins to labeled 3'-terminal RNA by mobility shift analysis. UV crosslinking revealed that the predominant 3'-terminal RNA-binding protein migrates as a single, 60-kDa species that can be precipitated by monoclonal antibodies directed against heterogeneous nuclear ribonucleoprotein I, also called polypyrimidine tract-binding protein (hnRNP-I/PTB), a protein previously shown to bind to the 5' internal ribosome entry site (IRES) of the HCV genome. Purified hnRNP-I/PTB also bound selectively to the 3' end of the HCV genome. hnRNP-I/PTB binding requires the upstream two stem-loop structures (SL2 and SL3) but not the most 3'-terminal stem-loop (SL1). Minor alteration of either the stem or loop sequences in SL2 or SL3 severely compromised hnRNP-I/PTB binding, suggesting extremely tight RNA structural requirements for interaction with this protein. hnRNP-I/PTB does not bind to either end of the antigenomic RNA strand and binds to the 5' IRES element of the genome at least 10-fold less avidly than to the 3' terminus. The strong, selective, and preferential binding of hnRNP-I/PTB to the 3' end of the HCV genome suggests that it may be recruited to participate in viral replication, helping to direct initiation of negative-strand RNA synthesis, stabilize the viral genome, and/or regulate encapsidation of genomic RNA.     

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.