KSHV Rta Promoter Specification and Viral Reactivation

Abstract

Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi's sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic "CANT DNA repeats" in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta's role as the switch is inefficient. Many factors modulate K-Rta's function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.

Keywords: DNA binding; DNA/protein interactions; KSHV; RBP-Jk; Rta; herpesvirus; reactivation.

Figure 1

K-Rta structure/function map. A linear representation of the amino acid content and predicted structural motifs of the ORF 50/Rta protein. Numbers refer to amino acid (AA) positions. Positions of each functional domain are shown by the colored bars, with amino acid boundaries indicated by numbers, corresponding to the function shown in the column on the right. Color codes for bars are: black, core functional domain; red, inhibitor of K-Rta; green, stimulator of K-Rta. Citations are listed in parentheses, and described in the text. +++, basic amino acid rich; LR, leucine heptapeptide repeat; ST, serine/threonine-rich; hyd/DE/hyd, repeats of hydrophobic and acidic amino acids, comprising K-Rta’s transcriptional activation domain; NLS, nuclear localization sequence; Dom. Neg., dominant negative.

Figure 2

Comparison of RBP-Jk independent and dependent K-Rta responsive elements. Vertical lines indicate homologous bases. Numbers indicate positions relative to transcriptional start sites. The green box indicates position of RBP-Jk binding site.

Figure 3

Comparison of consensus K-Rta binding sites. The indicated K-Rta consensus sequences were derived from the listed studies. “N” means any nucleotide.

Figure 4

Comparison of Mta and K-ZIP promoters. (A). Schematic of the −136/−62 Mta promoter. Sequences of the top and bottom strands of the indicated portion of the Mta promoter. Numbers indicate positions relative to Mta transcriptional start site. Bold letters indicate four units of the A/T₃ trinucleotide repeat. Underlines indicate Rta’s footprint from the bottom strand. Blue boxes indicate regions of highest conservation, nt 4–14, for each unit of the CANT repeat. The green box indicates the RBP-Jk binding site. The peach box indicates the AP-1 binding site. The yellow box indicates the K-RBP binding site. The red box indicates the IRF-7 binding site. The position of the short, Mta 5D element is indicated by the brackets. (B) Alignment of the top and bottom strands of the Mta and K-bZIP promoters. Numbering and box designations are as listed in the legend for Figure 3, above. The purple box indicates the Oct-1 binding site.

Figure 5

Model for stimulation of DNA binding of RBP-Jk by Rta. The sequence and description of the Mta promoter are as described in the legend for Figure 4A. Tetramerization of Rta allows it to straddle RBP-Jk and contact the palindromic CANT repeat units on both sides of RBP-Jk. A second K-Rta tetramer may bind to an unidentified protein (“X”) and contact the CANT repeats in the upstream side of the promoter. The two Rta tetramers might contact each other to stabilize the holo-protein/DNA complex.

Figure 6

Single cell model for progression of KSHV reactivation and its putative influence on cellular growth. As described in text, Rta expressing PEL cells are depicted as having three fates: complete reactivation (top), expression of a sub-set of DE genes without complete reactivation (middle), and completely abortive reactivation, in which K-Rta might feedback to activate itself (bottom). Modulators of K-Rta function that were discussed in the text are listed in the light blue boxes. Negative regulators (red text) might favor the non-productive reactivation fates, while positive regulators (green text) might favor the productive reactivation fate. DE oncogenes expressed in the top or middle fates, might cooperate with latent proteins to stimulate cell growth (red lines and text). IE, immediate early; DE, delayed early; vDNA repl, viral DNA replication.