Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP).Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.

We report combined experimental and theoretical investigations of x-ray absorption at the Ru-L 2,3 and O-K thresholds of the Ru͑IV͒ compounds RuO 2 and Sr 2 RuO 4 and of the Ru͑V͒ compound Sr 4 Ru 2 O 9 . Significant differences in the intensity distribution of the t 2g -related and e g -related peaks between the L 3 and the L 2 edges are found, due to the combined effects of 4d spin-orbit coupling and the interelectronic Coulomb interaction described by the Slater integrals. The observed spectral features can be well reproduced by crystal-fieldmultiplet calculations. With increasing the Ru valence from IV to V, the spectra are shifted by Х1.5 eV to higher energy at the Ru-L 2,3 edges and Х1.0 eV to lower energy at the O-K edge, which is of the same order of magnitude as on going from the divalent to the trivalent late 3d transition-metal oxides.

The Epstein-Barr virus (EBV) alkaline exonuclease BGLF5 has previously been recognized to contribute to immune evasion by downregulating production of HLA molecules during virus replication. We have constructed a BGLF5-null virus mutant to determine BGLF5's functions during EBV viral replication. Quantification of virus production in permissive 293 cells carrying a ⌬BGLF5 genome identified a 17-to 21-fold reduction relative to complemented or wild-type controls. Detailed monitoring of ⌬BGLF5 replication evidenced an impaired virus nucleocapsid maturation, a reduced primary egress and a 1.4-fold reduction in total viral DNA synthesis. ⌬BGLF5 single-unit-length viral genomes were not only less abundant but also migrated faster than expected in gel electrophoresis. We concluded that BGLF5 pertained both to the generation and to the processing of viral linear genomes. ⌬BGLF5 phenotypic traits were reminiscent of those previously identified in a mutant devoid of UL12, BGLF5's homolog in herpes simplex virus type 1, and indeed UL12 was found to partially complement the ⌬BGLF5 phenotype. However, BGLF5-specific functions could also be identified; the nuclear membrane of replicating cells displayed images of reduplication and complex folding that could be completely corrected by BGLF5 but not UL12. Similar nuclear abnormalities were previously observed in cells transfected with BFLF2 and BFRF1, two viral proteins crucial for EBV nuclear egress. Interestingly, ⌬BGLF5 cells produced more BFLF2 than wild-type or complemented counterparts. The present study provides an overview of BGLF5's functions that will guide future molecular studies. We anticipate that the 293/⌬BGLF5 cell line will be instrumental in such developments.The Epstein-Barr virus (EBV) is a predominantly B lymphotropic member of the gammaherpesvirus subfamily whose host spectrum is physiologically restricted to humans (35). EBV possesses a large genome that encodes for more than 100 genes, the majority of which are required for efficient virus replication and propagation. Although strictly dependent on its host cells for replication, EBV encodes several proteins endowed with enzymatic activities. Some enzymes, such as the viral DNA polymerase (Pol) BALF5, are directly involved in virus construction, but others interact with the cellular host (21). One example is provided by viral proteins, first identified in the alphaherpesviruses, that serve a host shutoff function (HSO), i.e., act as negative regulators of cellular protein production to the benefit of the virus. In herpes simplex virus (HSV), HSO leads to preferential synthesis of viral proteins and to downregulation of cellular proteins crucial for immune response against the virus (for a review, see reference 14). Previous genetic and biochemical studies have identified the UL41 gene product vhs (for viral host shutoff) as one essential mediator of HSV-1-induced HSO (24, 30); vhs is thought to curb cellular protein production through its RNase activity (6,23,41,45). More recent work has shown that this...