Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/ NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.Transcription in eukaryotic cells is a complex process and involves three major steps including initiation, elongation, and termination. Although it was thought originally that regulation occurs primarily at the level of initiation, it is now recognized that the elongation step of transcription is a critical target for regulation of gene expression (1-3). Density analysis of RNA pol 1 II in cells led to the identification of a number of genes including c-myc (4), c-fms (5), hsp70 (6), and HIV (7), which are potentially regulated at the elongation stage of transcription. Shortly after initiation, RNA pol II faces a barrier of negative transcription elongation factors and enters abortive elongation. The action of positive transcription elongation factors (P-TEF) lowers the barrier of negative transcription elongation factors and helps RNA pol II to escape from this transition phase which could lead to premature termination of transcription (for an excellent review, see Ref. 8). A positive elongation factor, P-TEFb, allows the transition into productive elongation producing longer mRNA transcripts (8).Proteins involved in positive and negative regulation of elongation were discovered during studies aimed at understanding the mechanism of transcription inhibition by a nucleoside analog, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). DRB was discovered as an inhibitor of hnRNA and mRNA synthesis in HeLa cells (9, 10). DRB inhibits the production of full-length RNA and increases the amount of short transcripts from a variety of genes, suggesting that RNA pol II elongation was affected (10). In addition, DRB has no effect on promoterindependent RNA pol II transcription and on transcription reconstituted by purified general transcription factors and...

Background. A unique genomic difference between human and civet severe acute respiratory syndrome coronaviruses (SARS-CoVs) is that the former has a deletion of 29 nucleotides from open reading frame (orf) 8a that results in the generation of orf8a and orf8b. The objectives of the present study were to analyze antibody reactivity to ORF8a in patients with SARS and to elucidate the function of ORF8a.Methods. Western-blot and immunofluorescent antibody assays were used to detect anti-ORF8a antibody. SARS-CoV HKU39849 was used to infect stable clones expressing ORF8a and cells transfected with small interfering RNA (siRNA). The virus loads (VLs) and cytopathic effects (CPEs) were recorded. Confocal microscopy and several mitochondria-related tests were used to study the function of ORF8a.Results. Two (5.4%) of 37 patients with SARS had anti-ORF8a antibodies. The VLs in the stable clones expressing ORF8a were significantly higher than those in control subjects 5 days after infection. siRNA against orf8a significantly reduced VLs and interrupted the CPE. ORF8a was found to be localized in mitochondria, and overexpression resulted in increases in mitochondrial transmembrane potential, reactive oxygen species production, caspase 3 activity, and cellular apoptosis.Conclusions. ORF8a not only enhances viral replication but also induces apoptosis through a mitochondriadependent pathway.

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