The alphaherpesvirus serine/threonine kinase us3 disrupts promyelocytic leukemia protein nuclear bodies

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection, including preventing virus-induced apoptosis, facilitating nuclear egress of capsids, stimulating mRNA translation and promoting cell-to-cell spread of virus infection. Given this diversity, the full spectrum of Us3 function may not yet be recognized. We noted, in transiently transfected cells, that herpes simplex virus type 2 (HSV-2) Us3 disrupted promyelocytic leukemia protein nuclear bodies (PML-NBs). However, PML-NB disruption was not observed in cells expressing catalytically inactive HSV-2 Us3. Analysis of PML-NBs in Vero cells transfected with pseudorabies virus (PRV) Us3 and those in Vero cells infected with Us3-null or -repaired PRV strains indicated that PRV Us3 expression also leads to the disruption of PML-NBs. While loss of PML-NBs in response to Us3 expression was prevented by the proteasome inhibitor MG132, Us3-mediated degradation of PML was not observed in infected cells or in transfected cells expressing enhanced green fluorescent protein (EGFP)-tagged PML isoform IV. These findings demonstrate that Us3 orthologues derived from distantly related alphaherpesviruses cause a disruption of PML-NBs in a kinase- and proteasome-dependent manner but, unlike the alphaherpesvirus ICP0 orthologues, do not target PML for degradation.     

Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11

Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication. Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function. HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II. The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11. Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11. The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML. Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin. These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication.     

Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies

The promyelocytic leukaemia (PML) tumour suppressor protein epitomizes the PML-nuclear body (PML-NB) and is crucially required for the proper assembly of this macromolecular nuclear structure. Unlike other, more specialized subnuclear structures such as Cajal and Polycomb group bodies, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions. PML-NBs are dynamic structures that favour the sequestration and release of proteins, mediate their post-translational modifications and promote specific nuclear events in response to various cellular stresses. Recent data suggest that PML-NBs may be heterogeneous in composition, mobility and function.     

Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.     

The cell biology of disease: Acute promyelocytic leukemia, arsenic, and PML bodies

Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation.