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Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication.

ABSTRACT:

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Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates : EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein.

Importance

Ebolavirus (EBOV) is one of the most lethal human pathogens known. EBOV requires host factors for replication due to its small RNA genome. Here we show that the host protein eIF5A in its activated form is necessary for EBOV replication. We further show that the mechanism is through the accumulation of a single EBOV protein, VP30. To date, no other host proteins have been shown to interfere with the translation or stability of an EBOV protein. Activated eIF5A is the only protein in the cell known to contain the specific modification of hypusine; therefore, this pathway is a target for drug development. Further investigation into the mechanism of eIF5A interaction with VP30 could provide insight into therapeutics to combat EBOV.

SUBMITTER: Olsen ME

PROVIDER: S-EPMC4981715 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Publications

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication.

Olsen Michelle E ME Filone Claire Marie CM Rozelle Dan D Mire Chad E CE Agans Krystle N KN Hensley Lisa L Connor John H JH

mBio 20160726 4

<h4>Unlabelled</h4>Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates : EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule in ...[more]

PMID: 27460797

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Project description:UNLABELLED:Recent work demonstrated that the Niemann-Pick C1 (NPC1) protein is an essential entry receptor for filoviruses. While previous studies focused on filovirus entry requirements of NPC1 in vitro, its roles in filovirus replication and pathogenesis in vivo remain unclear. Here, we evaluated the importance of NPC1, and its partner in cholesterol transport, NPC2, by using a mouse model of Ebolavirus (EBOV) disease. We found that, whereas wild-type mice had high viral loads and succumbed to EBOV infection, Npc1(-/-) mice were entirely free of viral replication and completely protected from EBOV disease. Interestingly, Npc1(+/-) mice transiently developed high levels of viremia, but were nevertheless substantially protected from EBOV challenge. We also found Npc2(-/-) mice to be fully susceptible to EBOV infection, while Npc1(-/-) mice treated to deplete stored lysosomal cholesterol remained completely resistant to EBOV infection. These results provide mechanistic evidence that NPC1 is directly required for EBOV infection in vivo, with little or no role for NPC1/NPC2-dependent cholesterol transport. Finally, we assessed the in vivo antiviral efficacies of three compounds known to inhibit NPC1 function or NPC1-glycoprotein binding in vitro. Two compounds reduced viral titers in vivo and provided a modest, albeit not statistically significant, degree of protection. Taken together, our results show that NPC1 is critical for replication and pathogenesis in animals and is a bona fide target for development of antifilovirus therapeutics. Additionally, our findings with Npc1(+/-) mice raise the possibility that individuals heterozygous for NPC1 may have a survival advantage in the face of EBOV infection. IMPORTANCE:Researchers have been searching for an essential filovirus receptor for decades, and numerous candidate receptors have been proposed. However, none of the proposed candidate receptors has proven essential in all in vitro scenarios, nor have they proven essential when evaluated using animal models. In this report, we provide the first example of a knockout mouse that is completely refractory to EBOV infection, replication, and disease. The findings detailed here provide the first critical in vivo data illustrating the absolute requirement of NPC1 for filovirus infection in mice. Our work establishes NPC1 as a legitimate target for the development of anti-EBOV therapeutics. However, the limited success of available NPC1 inhibitors to protect mice from EBOV challenge highlights the need for new molecules or approaches to target NPC1 in vivo.

Dataset Information

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication.

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Importance

Publications

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication.

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