Research Article: Progressive Accumulation of Activated ERK2 within Highly Stable ORF45-Containing Nuclear Complexes Promotes Lytic Gammaherpesvirus Infection

Date Published: April 10, 2014

Publisher: Public Library of Science

Author(s): Evonne N. Woodson, Melissa S. Anderson, Matthew S. Loftus, Dean H. Kedes, Fanxiu Zhu.

http://doi.org/10.1371/journal.ppat.1004066

Abstract

De novo infection with the gammaherpesvirus Rhesus monkey rhadinovirus (RRV), a close homolog of the human oncogenic pathogen, Kaposi’s sarcoma-associated herpesvirus (KSHV), led to persistent activation of the MEK/ERK pathway and increasing nuclear accumulation of pERK2 complexed with the RRV protein, ORF45 (R45) and cellular RSK. We have previously shown that both lytic gene expression and virion production are dependent on the activation of ERK [1]. Using confocal microscopy, sequential pull-down assays and FRET analyses, we have demonstrated that pERK2-R45-RSK2 complexes were restricted to the nucleus but that the activated ERK retained its ability to phosphorylate nuclear substrates throughout infection. Furthermore, even with pharmacologic inhibition of MEK beginning at 48 h p.i., pERK2 but not pERK1, remained elevated for at least 10 h, showing first order decay and a half-life of nearly 3 hours. Transfection of rhesus fibroblasts with R45 alone also led to the accumulation of nuclear pERK2 and addition of exogenous RSK augmented this effect. However, knock down of RSK during bona fide RRV infection had little to no effect on pERK2 accumulation or virion production. The cytoplasmic pools of pERK showed no co-localization with either RSK or R45 but activation of pERK downstream targets in this compartment was evident throughout infection. Together, these observations suggest a model in which R45 interacts with pERK2 to promote its nuclear accumulation, thereby promoting lytic viral gene expression while also preserving persistent and robust activation of both nuclear and cytoplasmic ERK targets.

Partial Text

The life cycle of all herpesviruses has two distinct phases: lytic and latent. During lytic infection, nearly the entire genome is undergoing active transcription. In contrast, during latency, gene expression is limited to a few genes and, in most species, miRNAs [2]–[4]. The gammaherpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV), the causative agent of three human malignancies, strongly favors latency following initial entry into a variety of target cells in culture [5]–[9]. Much of our current understanding ofthe lytic phase in KSHV derives from studies on the reactivation of the virus from latently infected primary effusion lymphoma (PEL) cell lines. In these patient derived cell lines, the addition of reactivating agents such as phorbol esters or HDAC inhibitors results in reactivation (productive infection) in less than 25% of the cells, making the study of lytic replication challenging. In contrast, the nonhuman primate homolog, Rhesus monkey rhadinovirus (RRV) that closely resembles KSHV in both sequence and genomic structural organization, displays a robust lytic infection following de novo infection of cultured rhesus fibroblasts (RhF) [10]. This allows the study of lytic infection and its regulationwithout the addition of exogenous inducing agents or overexpression of early components of the lytic cascade that can confound results.

One mechanism viruses use to promote their own propagation within host cells is the manipulation of signaling cascades, particularly the mitogen activated-protein kinase (MAPK) pathways [12]–[14], [20], [28], [68]. We previously reported elevated levels of phosphorylated ERK as late as 48 hours post-infection, though those studies examined the roles of ERK isoforms in RRV infection and virion assembly [1]. In the current study, we focused on the effects of sustained ERK activation as well as the mechanism underlying this activation during de novo RRV infection. We found that pERK levels increase minimally as early as 30 minutes post-infection and then drop by 2.5 h before beginning a continual rise until cell lysis, 48–72 h p.i. Sharma-Walia et al. showed that the addition of soluble KSHV viral envelope glycoprotein B (gB) to cultured fibroblasts or endothelial cells results in a rapid, but transient induction of ERK phosphorylation. However, we observed a similar initial transient increase in pERK in UI as well as UV-RRV infected cells at 30 minutes, suggesting that, in our experimental system, any initial increase in pERK levels due to virus binding is likely masked by the stimulation from the addition of fresh serum-containing media since that step was common among all three experimental conditions and serum can stimulate a rapid and transient induction of ERK phosphorylation [69].

 

Source:

http://doi.org/10.1371/journal.ppat.1004066

 

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