The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb C(term)) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb C(term) phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4-E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

Latent Kaposi's sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.

The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.