Distinct DNA targets for trans-activation by HTLV-1 tax and adenovirus E1A.

The HTLV-1 LTR is trans-activated by both the HTLV-1 tax (p40x) and adenovirus E1A gene products. Previous experiments have localized tax-responsive cis-elements to three 21-bp repeat units within the promoter, as well as a fourth region located between the middle and proximal repeats. A sequence TGACG, resembling the ATF/CREB recognition element, is located at the center of each of these repeat units. Mutation of this ATF/CREB site in the 21-bp repeats impairs both tax and E1A-dependent trans-activation. However, assays of a variety of promoter mutants demonstrate that sequences required for E1A and tax induction differ, suggesting that these two viral trans-activators target different factors. In addition, although the adenovirus E4 promoter also contains three ATF/CREB sites involved in E1A activation, tax does not activate this promoter. Finally, we also find that the TATAA element of the HTLV-1 LTR contributes to E1A-dependent activation but not tax activation. We concluded that although both trans-activators exhibit similarities in their activation properties, the targets for activation must differ.     

A combination of defective DNA and protective host factors are found in a set of HIV-1 ancestral LTNPs

We studied viral evolution in three HIV-1 ancestral patients from a group of LTNPs; although some minor sequences showing viral evolution were detected in all patients, the extremely low viral evolution of their viruses was shown by the phylogenetic analysis of the env sequences. Complete nucleotide sequencing of viral DNA showed the major presence of deletions. In two patients, deletions of 1088 and 228 nucleotides mapped to 5′ LTR-gag region; in the other, a 247 nucleotide deletion was positioned in pol gene up to the vif ORF. These deleted genomes became dominant during follow up. Patient's viruses displayed 13 common mutations in conserved residues, from the 5′ LTR to the nef gene. These mutations provided evidence of a common origin. Regarding host characteristics, one patient had HLA B2705/B5801; another B1402/B5701; whereas a third showed B3901/B4402 and was Δ32-CCR5 heterozygous. These HIV controllers presented a combination of deleted viral genomes and host protective factors.     

Deletion Analysis of Both the Long Terminal Repeat and the Internal Promoters of the Human Foamy Virus

Deletion analyses of the long terminal repeat (LTR) and internal promoters (IP) of human foamy virus (HFV) showed that a negative acting element resides in the U5 region of the 5′ LTR reducing reporter gene expression tenfold. The basal activity of the IP was higher than that obtained with LTR promoter constructs and strongly elevated in permissive BHK-21 cells whereas semi-permissive COS-7 cells showed low basal activity. Since the basal activity of the IP is critical for initiating HFV gene expression by providing Bel 1 transactivator early after infection, the basal activity of the IP may be the crucial factor that contributes to whether cells are permissive for HFV infection or not. Deletion mutagenesis allowed to define the minimal IP region. A region strongly transactivated by Bel 1 extends from −136 to +58 relative to the cap site of the IP. The major Bel 1 response element of the IP required for transactivation is located upstream of the cap site between −136 and −88 relative to the internal cap site. A DNA fragment reported to be protected by recombinant Bel 1 was deleted with marginal reduction of Bel 1 transactivation. HFV gene expression directed by the IP and LTR promoters is thus multiply regulated by positive and negative acting response elements in cis and their binding partners in trans. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Fimbria Gene Cluster of Nonencapsulated Haemophilus influenzae

The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the −10 and −35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.     

Replication of an association of a promoter polymorphism of the dopamine transporter gene and Attention Deficit Hyperactivity Disorder

Genetic associations for Attention Deficit Hyperactivity Disorder (ADHD), a common highly heritable childhood behavioural disorder, require replication in order to establish whether they are true positive findings. The current study aims to replicate recent association findings from the International Multi-centre ADHD Genetics (IMAGE) project in one of the most studied genes related to ADHD, the dopamine transporter (DAT1) gene. In a family-based sample of 450 ADHD probands, three Single Nucleotide Polymorphism (SNP) markers have been genotyped using TaqMan assays. Transmission Disequilibrium Test analysis demonstrates that one of three SNP markers (rs11564750) in the 5′ promoter region of the gene is significantly associated with ADHD (P = 0.02). This provides further evidence that in addition to the well-known and investigated 3′UTR polymorphism associated with ADHD, there is potentially a further association signal emanating from the 5′ promoter region of the gene. Further replication and functional studies are now required to fully understand the consequence of polymorphisms present at both the 5′ and 3′ ends of the DAT1 gene and their role in ADHD pathophysiology.     

Induction of Tax i expression in MT-4 cells by 5-azacytidine leads to protein binding in the HTLV-1 LTR in vivo

The Tax I trans-activator protein of the human T-cell leukemia virus I (HTLV-I) enhances viral gene expression through enhancer sequences in the viral LTR. These sequences consist of three imperfect 21-bp repeats (TRE-1) and a region between the promoter central and promoter proximal TRE-1 (known as TRE-2). We have previously described the in vivo footprint of the HTLV-I TRE-1s and TRE-2 in two HTLV-I-infected cell lines, MT-2 and MT-4. MT-2 is a high-level producer of virus and shows significant DNA-protein interactions within the TRE-1s and TRE-2. In contrast, the proviral DNA in MT-4 cells is heavily methylated and produces no detectable virus. In this report, we describe the footprints of the TRE-1s and TRE-2 in MT-4 cells that were induced to express high levels of viral proteins by treatment with 5-azacytidine, a potent inhibitor of methylation. The footprints of the TRE-1s in 5-azacytidine-treated MT-4 cells were virtually identical to those observed in MT-2 cells. In contrast, the footprints within the TRE-2 region of 5-azacytidine-treated MT-4 cells did not resemble those in either MT-2 or MT-4 cells.     

Non-random deletions in human foamy viruslong terminal repeat during viral infection

Summary.  We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence. Received July 22, 1996 Accepted January 10, 1997