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Background. Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients.Methods. Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome.Results. Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement.Conclusions. These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.

Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the aral site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when aral and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are aral, araO2, and perhaps the araBAD operon RNA polymerase binding site.The L-arabinose operon of Escherichia coli has long been known to be positively regulated by AraC protein (1). Classical genetic (2-f: and biochemical (5-7) studies have also revealed that th;e) dRAD genes are negatively regulated by AraC and that a site required for this repression is located upstream from all the sites required for induction (2,8,10).Recently, a site required for repression, araO2, was found and was determined to lie 210 base pairs upstream from the AraC binding site required for induction, araI ( Fig. 1) (8). Surprisingly, the behaviors of strains with small deletions and insertions between the araBAD induction region and the araO2 site suggest that repression of the araBAD operon involves the formation of a DNA loop that brings araO2 near the induction region (8). Since repression of a constitutive araBAD promoter requires functional araO2 and araI sites as well as the presence of AraC protein (7) The results of genetic experiments were also as predicted by the DNA loop model. Mutations isolated solely on the basis that they reduced repression were found to lie in the araO2 and araI sites. However, repression-defective mutations also were found in the araBAD operon RNA polymerase binding site, raising the possibility of the involvement of RNA polymerase in repression. Mutations constructed in other known protein binding sites in the regulatory region did not interfere with repression. The results of these standard genetic experiments suggest that only the araI, araO2, and possibly RNA polymerase binding sites are involved in repression. These results strengthen previous results obtained by using "inverse genetics" in which regions of DNA are intentionally mutated and the results observed. MATERIALS AND METHODSMedia, Plasmids, Strains, and General Methods. Media and general methods were as described (11,12). Plasmid pTD3 (9) contains 440 base pairs of the araCBAD regulatory region on a HindIII/EcoRI fragment with PBAD driving galK of the pKO1 vector (13). The plasmid with the araCBAD induction region deleted and used for in vivo footprinting of araO2, pLH1, was made by deleting from to BamHI (position -46) of pTD3. AraO2 deletions of plasmids with araI ...