Inhibition of primary effusion lymphoma engraftment in SCID mice by morpholino oligomers against early lytic genes of Kaposi's sarcoma-associated herpesvirus.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells. METHODS: PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis. RESULTS: Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study. CONCLUSIONS: This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.

Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers.

Porcine reproductive and respiratory syndrome (PRRS) has caused heavy economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) can be an effective antiviral against the PRRS virus (PRRSV). PPMO is structurally similar to DNA with modified backbone and is resistant to nuclease. This study was designed to examine increasing inhibitory effect of PPMO combination. Two pairs of PPMOs were identified to have enhanced suppression of PRRSV replication in cell culture, while individual constituents did not work under the same testing conditions. PPMO 5UP1 that is complementary to 5' terminus of PRRSV genome was paired with 4P1 or 7P1 that are complementary to sequence in the translation initiation regions of ORFs 4 and 7, respectively. The PPMO combination also inhibited replication of heterologous strains in the North American PRRSV genotype. Treatment of the cells with the combinations reduced PRRSV RNA and protein levels. In cell-free or cell-based luciferase reporter assays, the PPMO combination suppressed target mRNA translation more effectively than individual constituents, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for PRRS control.

The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.

Suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a contagious disease characterized by reproductive failure in sows and respiratory disease in piglets. This infectious disease results in significant losses in the swine industry and specific anti-PRRSV drugs are needed. In this study, we evaluated a novel class of antisense compounds, peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), for their ability to suppress PRRSV replication in cell culture. P-PMOs are analogs of single-stranded DNA and contain a modified backbone that confers highly specific binding to RNA and resistance to nucleases. Of six P-PMOs tested, one ('5UP1'), with sequence complementary to the 5'-terminal 21 nucleotides of the PRRSV genome, was found to be highly effective at reducing PRRSV replication in a specific and dose-dependent manner in CRL11171 cells in culture. 5UP1 treatment generated up to a 4.5log reduction in infectious PRRSV yield, while a control P-PMO had no effect on viral titer. Immunofluorescence assay with an anti-PRRSV monoclonal antibody confirmed the titer observations. The sequence-specificity of 5UP1 effect was confirmed in part by a cell-free luciferase reporter assay system, which showed that 5UP1-mediated inhibition of translation decreased if the target-RNA contained mispairings in relation to the 5UP1 P-PMO. Real-time RT-PCR showed that the production of PRRSV negative-sense RNA was reduced if 5UP1 was added to cells at up to 6h post-virus inoculation. Cell viability assays detected no cytotoxicity of 5UP1 within the concentration-range of this study. These results indicate that P-PMO 5UP1 has potential as an anti-PRRSV agent.