Identification of RNA instability elements in Borna disease virus.

Genome organization and gene expression of Borna disease virus (BDV) are remarkable for the overlap of open reading frames, transcription units and transcription signals, readthrough of transcription termination signals, differential use of translation initiation codons, and exploitation of the cellular splicing machinery. Here we report an additional control of gene expression at the level of mRNA stability. Levels of BDV proteins in infected cells do not correspond to the transcriptional gradient typically observed in nonsegmented negative-sense RNA viruses. The third transcription unit of BDV's negative-sense RNA genome encodes viral proteins M, G and L. Analysis of the third transcription unit identified RNA-destabilizing domains with the most pronounced activity located in regions spanning nucleotides 2818-2918 (instability domain-1) and 4022-4071 (instability domain-2). Given that one domain maps to intron-2 and is thereby eliminated upon splicing, this represents an intriguing mechanism for regulating transcript levels independent of a transcriptional gradient. The presence of instability domains in introns offers a mechanism to create the observed discontinuous gradient M>L>G, compatible with the non-cytopathic, persistent infection that is characteristic for BDV, and provides a rationale for the use of alternative splicing by this unusual virus.

Lack of association between measles virus vaccine and autism with enteropathy: a case-control study.

BACKGROUND: The presence of measles virus (MV) RNA in bowel tissue from children with autism spectrum disorders (ASD) and gastrointestinal (GI) disturbances was reported in 1998. Subsequent investigations found no associations between MV exposure and ASD but did not test for the presence of MV RNA in bowel or focus on children with ASD and GI disturbances. Failure to replicate the original study design may contribute to continued public concern with respect to the safety of the measles, mumps, and rubella (MMR) vaccine. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this case-control study was to determine whether children with GI disturbances and autism are more likely than children with GI disturbances alone to have MV RNA and/or inflammation in bowel tissues and if autism and/or GI episode onset relate temporally to receipt of MMR. The sample was an age-matched group of US children undergoing clinically-indicated ileocolonoscopy. Ileal and cecal tissues from 25 children with autism and GI disturbances and 13 children with GI disturbances alone (controls) were evaluated by real-time reverse transcription (RT)-PCR for presence of MV RNA in three laboratories blinded to diagnosis, including one wherein the original findings suggesting a link between MV and ASD were reported. The temporal order of onset of GI episodes and autism relative to timing of MMR administration was examined. We found no differences between case and control groups in the presence of MV RNA in ileum and cecum. Results were consistent across the three laboratory sites. GI symptom and autism onset were unrelated to MMR timing. Eighty-eight percent of ASD cases had behavioral regression. CONCLUSIONS/SIGNIFICANCE: This study provides strong evidence against association of autism with persistent MV RNA in the GI tract or MMR exposure. Autism with GI disturbances is associated with elevated rates of regression in language or other skills and may represent an endophenotype distinct from other ASD.

Viral and nonviral factors causing nonspecific replication of tumor- and tissue-specific promoter-dependent oncolytic adenoviruses.

Restricted replication-competent adenoviruses (RRCAs) using tumor- and tissue-specific promoters (ttsP's) are new tools for cancer gene therapy. In this study we investigated viral and nonviral factors affecting "leakiness" of several ttsP's and their relevance for nonspecific ttsP-dependent RRCA (ttsP-RRCA) replication. The leakiness of the ttsP's in nontarget cells was per se highly variable and correlated with levels of nonspecific ttsP-RRCA replication. Transcriptional regulator elements fused to ttsP's showed variable effects: a hypoxic response element reduced leakiness of an alpha-fetoprotein promoter. In contrast, a mouse tyrosinase enhancer increased leakiness of a tyrosinase promoter, although it was not affected by a human tyrosinase enhancer. Furthermore, leakiness of ttsP's was enhanced by 5'-terminal adenoviral E1A enhancers, and adenoviral E1A-13S was found to be a strong transactivator of ttsP's, leading to "autoactivation" of leaky ttsP-RRCAs. In a proof-of-principle study, ttsP-RRCA replication was shown to be inhibited by a tetracycline-controlled transcriptional silencer via direct ttsP silencing. This opens up the prospect of pharmacological regulation of ttsP-RRCAs. Together, these data indicate that leakiness of ttsP's induced by several factors is a major cause of nonspecific ttsP-RRCA replication. Consideration of these factors may help optimize ttsP-dependent RRCA vectors and may thereby improve their safety.

Regulation of human factor IX expression using doxycycline-inducible gene expression system.

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.