Protein kinase inhibitors that inhibit induction of lytic program and replication of Epstein-Barr virus.

Signaling pathways mediating Epstein-Barr virus (EBV) reactivation by Ag-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporine, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48 h after reactivation perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, glycogen synthase kinase 3ß (GSK-3ß), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK, respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication.

Expression and localization of the Epstein-Barr virus-encoded protein kinase.

The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding to the N terminus of EBV PK. Based on previously published data (E. Gershburg and J. S. Pagano, J. Virol. 76:998-1003, 2002) and the expression pattern described here, we conclude that EBV PK is an early protein that requires viral-DNA replication for maximum expression. By biochemical fractionation, the protein could be detected mainly in the nuclear fraction 4 h after viral reactivation in Akata cells. Nuclear localization could be visualized by indirect immunofluorescence in HeLa cells transiently expressing EBV BGLF4 in the absence of other viral products. Transient expression of 3'-terminal deletion mutants of EBV BGLF4 resulted in cytoplasmic localization, confirming the presence of a nuclear localization site in the C-terminal region of the protein. In contrast to the wild-type EBV PK, all of the mutants were unable to hyperphosphorylate EA-D during coexpression or to phosphorylate ganciclovir, as measured by an in-cell activity assay. Thus, the results demonstrate that the nuclear localization, as well as the kinase activity, of BGFL4 is dependent on an intact C-terminal region.

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.

Baculovirus-mediated expression of a scorpion depressant toxin improves the insecticidal efficacy achieved with excitatory toxins.

The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti-insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild-type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.

Expression of the Autographa californica nuclear polyhedrosis virus apoptotic suppressor gene p35 in nonpermissive Spodoptera littoralis cells.

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.

The wild-type Autographa californica nuclear polyhedrosis virus induces apoptosis of Spodoptera littoralis cells.

Spodoptera littoralis cells infected with the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) yielded significantly lower budded virus titers than Spodoptera frugiperda-infected cells and produced very low levels of polyhedrin. Relative to AcMNPV-infected S. frugiperda SF9 cells viral DNA replication was severely reduced in Spodoptera littoralis SL2 cells. Microscopic examination of SL2-infected cells revealed progressive cell blebbing starting at 6-8 hr postinfection and culminating in total cell destruction at 24 hr postinfection. The data suggested that AcMNPV-infected SL2 cells undergo apoptosis. The occurrence of an active apoptotic process in the infected cells was confirmed by: (1) observation of fragmentation of the cell nuclei stained with the specific fluorescent dye DAPI (4',6'-diamidino-2-phenylindole) and (2) the presence of low-molecular-weight DNA oligomers. Neither SL2 cells infected with S. littoralis nuclear polyhedrosis virus (SINPV) nor SF9 cells infected with AcMNPV, respectively, showed nuclear fragmentation or oligonucleosomal ladder formation.