Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

“…Work carried out by the Glazer group used bis-PNAs to target several polypurine sequences of the b-thalassemia mutation. Correction frequencies of up to 0.4 % for the splice-site mutation IVS2-1 were recorded by using a combination of bis-PNA and chloroquine treatment [54] NMA/2'-MOE PTEN [56] PMO LANA [57] HIV-1 PNA TAR [58] PNA PBS [59] LNA multiple [60] E. coli PNA and PMO acpP [61,62] PNA 23S rRNA [63] PNA and LNA RNase P [64] SARS-CoV PMO multiple [65] HCV PNA IRES [66] 2'-OMe E1 RNA [67] BMD/DMD PMO dystrophin gene [68,69] alphavirus PMO AUG translation [55] prions PS prion protein [70,71] b-thalassemia PNA IVS2-1 [72] AT PMO ATM [73] influenza A PMO PB1 and -2, NP [74,75] hypercholesterolemia LNA miR-122 [76] ChemBioChem www.chembiochem.org during the cells' S phase. Furthermore, sequences targeted with distances of several hundred base pairs away from the mutation still showed activity; this shows that further understanding of the mechanism of triplex stimulated recombination is still required.…”

Enhancement of Interferon Induction by ORF3 Product of Hepatitis E Virus

“…Whole-cell lysates were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting as described previously (15,30). Antibodies against GFP (Santa Cruz Biotechnology, Santa Cruz, CA), FLAG (Sigma, St. Louis, MO), hemagglutinin (HA; Thermo Fisher Scientific, Waltham, MA), Myc (Rockland Immunochemicals, Inc., Gilbertsville, PA), vp13, RIG-I (Santa Cruz), and tubulin (Sigma) were used for blotting.…”

Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Contributes to Viral Latent Replication by Activating Phosphorylation of Survivin

“…LANA, encoded by open reading frame 73 (ORF73), is the major latent protein expressed in all forms of KSHV-associated malignancies. LANA is a multifunctional protein which has the ability to (i) associate with cellular chromatin to maintain the viral episome (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), (ii) activate or repress transcription (9,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), (iii) subvert tumor suppressors (38,40,(43)(44)(45), (iv) stimulate cellular transformation (16,34,(46)(47)(48)(49)(50)(51), and (v) block apoptosis (9,49,(52)(53)(54)…”