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Lack of anin vitroculture system for human hepatitis B virus has hampered the ability to address fundamental questions regarding the viral life cycle and the effect of viral gene products during productive infection. To study the activity of HBV X protein (HBx) in the context of a viral infectious cycle, we provided HBxin transduring adenovirus infection of liver-derived cells. In hepatoma cells infected with adenovirus mutants deficient in expression of various E1A products, HBx was able to partially substitute for the transcriptional activation function of E1A. HBx also activated adenovirus replication, but to a lesser extent than the activation of transcription. Adenovirus genes transcribed by either RNA polymerase II or RNA polymerase III were activated by HBx during infection. These results suggest that HBx and E1A activate transcription by a similar mechanism and that this viral infection system will be useful for characterization of the functional activities of HBx.

Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX. The minimal region in pX sufficient for its interaction with TBP includes aa 110-143. Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX. The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP. These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters. This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.

BACKGROUND Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging cause of disease with serious potential consequences in children. The epidemiology and clinical spectrum of O26:H11 are incompletely understood. We investigated an outbreak of O26:H11 infection among children younger than 48 months of age and employees at a child care center. METHODS Every employee at the center (n = 20) and every child

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liver-specific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAACAGTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.

The human hepatitis B virus (HBV) is a member of the hepadnavirus family, which is compromised of DNA viruses that share structural and biological features. HBV infectivity is characterized by a marked hepatotropism, which is a prominent feature of hepadnaviruses. Although the majority of individuals infected with HBV resolve the primary infection and develop lasting immunity to subsequent infection, clinical studies have shown that 5–10% of individuals are chronically infected with the virus (Redeker, 1975). It has been estimated that there are over 250 million chronic HBV carriers worldwide (Rossner, 1992). A clinical consequence of chronic HBV infection is cirrhosis of the liver, which can lead to liver failure and death (Degroote et al., 1979). Furthermore, epidemiological studies have demonstrated that there is a strong association between hepatocellular carcinoma (HCC) and chronic HBV infection (Szmuness, 1978; Beasley et al., 1981). This association is supported by the following evidence: 1) there is a strong correlation in the geographical distribution of HCC and the occurence of chronic HBV infection, and 2) the hepatitis B surface antigen (HBsAg) is detected with a marked frequency in individuals with HCC. There is additional compelling evidence which correlates HBV with carcinogenesis.

The hepatitis B virus (HBV) X gene product trans-activates viral and cellular genes. The X protein (pX) does not bind independently to nucleic acids. The data presented here demonstrate that pX entered into a protein-protein complex with the cellular transcriptional factors CREB and ATF-2 and altered their DNA binding specificities. Although CREB and ATF-2 alone did not bind to the HBV enhancer element, a pX-CREB or pX-ATF-2 complex did bind to the HBV enhancer. Thus, the ability of pX to interact with cellular factors broadened the DNA binding specificity of these regulatory proteins and provides a mechanism for pX to participate in transcriptional regulation. This strategy of altered binding specificity may modify the repertoire of genes that can be regulated by transcriptional factors during viral infection.

Cytoplasmic RNA was isolated from varicella-zoster virus (VZV)-infected cells. By oligo(dT)-cellulose chromatography, the RNA was separated into polyadenylated, poly (A)+, and nonpolyadenylated, poly (A)-, fractions. RNA blot hybridization was employed to detect and map VZV transcripts. As VZV infection cannot be coordinated, cytoplasmic RNA was isolated from VZV-infected cells when the cells showed extensive cytopathology. Therefore, while the VZV transcripts represented heterogeneous temporal classes, it may be assumed that late VZV RNA predominated. At least 41, and as many as 67 (depending on DNA probe overlap), VZV polyadenylated transcripts have been identified. Preliminary evidence for the presence of two VZV-specific nonpolyadenylated, cytoplasmic transcripts was observed.