KSHV Rta Promoter Specification and Viral Reactivation

The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-␣/␤R ؊/؊ mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-␣). 5= rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-␣ treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-␣/␤R

Section: Characterization Of the Orf50 Distal Promoter Activity In VImentioning

confidence: 88%

Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

Wakeman¹,

Johnson²,

Paden³

et al. 2014

“…The KSHV ORF50-encoded immediate early protein RTA is known to activate the cascade of lytic genes for facilitating the lytic replication process (38)(39)(40)(41)(42). Studies on the primary infection of B cells by EBV showed an accumulation of lytic transcripts during infection, indicating the requirement of a short lytic replication prior to the establishment of latency (21,23).…”

Section: Discussionmentioning

confidence: 99%

“…The KSHV latency-associated protein ORF73 is the predominant protein expressed during latency and is responsible for immune evasion, latent DNA replication, and genome maintenance, whereas the ORF50-encoded protein RTA activates lytic cycle-specific KSHV genes in a cascaded manner to facilitate the lytic replication process (38)(39)(40)(41)(42). In a cell culture system, the overexpression of RTA is capable of triggering the lytic DNA replication (38,42). Our data detecting the transcripts of ORF50 and other lytic genes along with ORF73 during early infection suggest that KSHV may enter into a DNA replicative phase before establishing latent infection.…”

Section: Kshv Viral Transcripts Are Abundantly Present During the Primentioning

confidence: 99%

Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

Purushothaman

Thakker

Verma

2015

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), is a double-stranded DNA virus that causes Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease (1-3). Like other herpesviruses, KSHV exhibits both latent and lytic modes of infection, persisting predominantly in the latent state in which only a subset of the viral proteins are expressed, including the latency-associated nuclear antigen (LANA) protein (4-8). Although the expression of latent proteins plays a critical role in inducing and maintaining KSHV latency, the infected cells are primed early during the primary infection to retain the viral genomes and induce tumors (9). During the primary infection, KSHV undergoes a short lytic replication cycle that transcribes an array of viral genes, which have been shown to modulate various pathways for establishing the latent infection (9). In addition, a small fraction (1 to 5%) of the infected cells spontaneously undergo lytic reactivation to produce infectious virions, which is likely to be essential for increasing the population of infected cells and inducing viral pathogenesis (10-13).The infection of target cells with KSHV is a complex multistep process involving a variety of host cell surface receptors and multiple viral glycoproteins. Irrespective of its mechanism of entry, for a successful infection, KSHV must overcome the obstacles it encounters during the transportation of viral capsids from the plasma membrane into the nucleus. The main obstacles include

“…In this scenario, Rta production would increase phosphorylation of a very small amount of Pin1, which could then bind to Rta and exert its downstream effects. We cannot exclude the possibility that Pin1 also regulates Rta indirectly, however, through proteins that enhance or repress Rta function (56).…”

Section: Discussionmentioning

confidence: 99%

The Cellular Peptidyl-Prolyl cis / trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Guito

Gavina

Palmeri

et al. 2014

Self Cite

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that prolinedirected modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine-or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1's effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.