VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice

Abstract

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time-and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.The Filoviridae family consists of only two genera, Ebola virus (EBOV) and Marburg virus (MARV), and belongs to the order Mononegavirales. Filoviruses possess a nonsegmented, single-stranded RNA genome of negative polarity that contains seven genes. The nontranscribed genomic ends, termed "leader" (3Ј end) and "trailer" (5Ј end), harbor cis-acting signals important for viral transcription, replication, and encapsidation. Transcription and replication of the viral genome requires viral proteins (VP) VP30 and VP35, the nucleoprotein (NP), and viral polymerase L (27, 28). Along with its role in viral RNA synthesis, VP35 has also been shown to act as a type I interferon antagonist (2). As the only viral surface protein, the glycoprotein (GP) is involved in binding and entry of the virion into host cells. The matrix protein VP40 functions in virus assembly and budding, and it has been suggested that the minor matrix protein VP24 also plays a role in these processes (7,14).Most MARV and EBOV species cause a severe hemorrhagic fever associated with fatality rates up to 90% in humans and nonhuman primates (8, 32). Due to the high mortality rates and the lack of any approved effective human therapeutic or vaccine (5), filoviruses have been classified as biological safety level 4 (BSL-4) agents. Many approaches have been employed in attempts to develop effective therapies for EBOV, including the administration of nucleoside analogues (i.e., ribavirin), immune globulins, type I interferons and other cytokines, anticoagulants, and therapeutic vaccines (10,13,19,20,24,37,39,40). To date, th...

Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

“…Although the nature of the exact ligands that activate RLRs in vivo have not yet been definitively identified, dsRNA-bound RIG-I and MDA5 structures show that multiple PAMPs, including double-strandedness, dsRNA blunt ends, and short dsRNA with 5′OH or 5′PPP, can activate RLRs (34). A correlation between dsRNA-binding ability of zVP35 and its IFN-inhibitory effect on RLR function has been documented in several studies (15,16,19,21), and its biological relevance is supported by the observations that preactivation of RIG-I dramatically decreases ZEBOV yield and that recombinant ZEBOVs with mutations at critical RNAbinding residues are attenuated in vitro and avirulent in vivo (29,41). Our structural findings are consistent with the in vitro dsRNA-binding studies, suggesting that the crystal structure reflects a potentially physiologically relevant complex.…”

“…However, only zVP35 is able to inhibit dsRNA-mediated RLR activation by short dsRNAs in a dose-dependent manner. This correlates with the ability of zVP35 to compete with RIG-I for dsRNA backbone and blunt-end binding because mutation of residues such as F239, R312, R319, and K322 leads to a loss of dsRNA binding and a corresponding loss of RIG-I inhibition (15,16,19,21). We also show that both MARV and ZEBOV can effectively inhibit RLR activation by pI:C, presumably through interactions with the dsRNA backbone because the dominant PAMP in pI:C is the double-strandedness.…”

“…11 and 12). EBOV VP35 interacts with several components of innate antiviral defenses, including retinoic-acid inducible gene-I (RIG-I)-like receptor (RLR) pathways that lead to IFN production (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). These include inhibition of IFN production through double-stranded (ds)RNA sequestration and inhibition of IFN regulatory factor (IRF) 3/IRF7 phosphorylation by direct association with kinases that activate IRF3/IRF7, IKKe/TBK-1 (13,15,21,25).…”

Filovirus Replication and Transcription

“…Since filoviruses encode their own replication/transcription machinery, the components involved in replication/transcription; cis-acting elements on the genomes and VPs mediating replication/transcription, are ideal targets for antiviral strategies. Indeed, initial approaches to use viral genes and proteins involved in replication/transcription as targets for antiviral compounds were quite promising [59,63,[67][68][69][70]. Thus, a deeper understanding of the way in which filoviruses replicate and transcribe their genomes will lead to potential benefits in controlling filovirus infections.…”

Structure of the Ebola VP35 interferon inhibitory domain

“…Therefore, a functional VP35 is required for efficient viral replication and pathogenesis; knockdown of VP35 leads to reduced viral amplification and reduced lethality in infected mice (14)(15)(16)(17)(18). However, limited information is available regarding how VP35 is able to perform multiple functions.…”