Structural and Functional Analyses of Kaposi Sarcoma-associated Herpesvirus ORF57 Nuclear Localization Signals in Living Cells

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional, nuclear protein involved in post-transcriptional regulation of a subset of viral genes during lytic replication. Three nuclear localization signals (NLSs), NLS1 (amino acids (aa 101-107), NLS2 (aa 121-130), and NLS3 (aa 143-152), were identified in the N terminus of the ORF57 protein, and each of the three represents a short stretch of basic amino acid residues. Disruption of all three NLSs prevented localization of ORF57 in the nucleus. Insertion of individual NLSs into a heterologous cytoplasmic protein converted it into a nuclear protein, confirming that each NLS functions independently and is sufficient to promote protein nuclear localization. Although it exhibits a function similar to that of Epstein-Barr virus EB2 in promoting KSHV ORF59 expression, KSHV ORF57 differs from the herpes simplex virus ICP27 protein, and its function could be disrupted by point mutations of single or two NLSs in random combination, despite the proper localization of the mutant protein in the nucleus. The dysfunctional ORF57 containing NLS mutations also had low affinity with ORF59 RNA and the RNA export factor REF. However, the REF binding of ORF57 in vivo appeared to have no effect on ORF57-mediated enhancement of ORF59 expression. Thus, the three NLSs identified in ORF57 provide at least two functions, nuclear localization of ORF57 and up-regulation of ORF59 expression.

Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs

“…Examples include translation and mRNA stability, the nuclear egress of mRNA and mRNA stability, and the transcription and nuclear egress of mRNA. In looking at the literature on ORF57 action, it is often difficult to distinguish what is multifunctional activity from what is a single activity manifest in different, linked, functional processes (6,17,20,26,32). Similar difficulties can be seen in the data reported here.…”

“…The timing and intensity of viral gene expression is orchestrated by comprehensive, interrelated regulatory mechanisms. ORF57 is expressed in the immediate-early phase of lytic replication (20,24,28) and is a key orchestrator of the viral lytic cycle by inducing delayed early and late gene expression (15,20,25,26,32). The extremely low levels of expression of envelope glycoproteins until the final stage of lytic replication may serve in part to limit any cytotoxicity of virus-encoded glycoproteins and may also aid virus replication by limiting the exposure of these antigens to the host immune response.…”

“…KSHV ORF57 has been shown to enhance, by as-yet unknown mechanisms, mRNA levels of ORF59 and nut-1/polyadenylated nuclear (PAN) RNA (20,26). However, it has been difficult to demonstrate experimentally that this enhancement of mRNA accumulation is independent of nuclear export.…”

Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression

“…1). Although the cleaved ORF57 retains three intact NLSs (15), it remains mainly in the cytoplasm of infected cells. The consequence of caspase-7 cleavage of ORF57 in lytic KSHV infection is reducing a subset of viral lytic gene expression and virus production.…”

“…Plasmids-Plasmids pVM7 (KSHV ORF57 with a FLAG tag on its C terminus), pVM9 (KSHV ORF56 with FLAG tag on its C terminus), and pST3 (KSHV K8␤ cDNA) were described previously (15)(16)(17). Plasmid pVM52 expresses KSHV ORF57 fused to a FLAG tag on its N terminus.…”