Journal
JOURNAL OF VIROLOGY
Volume 76, Issue 10, Pages 5000-5013Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.76.10.5000-5013.2002
Keywords
-
Categories
Funding
- NCI NIH HHS [R01 CA091791, CA83525, CA91791] Funding Source: Medline
- NIDCR NIH HHS [DE14153, R01 DE014153] Funding Source: Medline
Ask authors/readers for more resources
RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpes-virus (KSM)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (K-d) of Rdbd on the pPAN RRE was determined to be approximately 8 X 10(-9) M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in Nitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available