Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis

AbstractEbola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30–eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30–eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.</jats:p

Ebola virus VP35 interaction with dynein LC8 regulates viral RNA synthesis

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8

Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication

Mutual Antagonism between the Ebola Virus VP35 Protein and the RIG-I Activator PACT Determines Infection Outcome

SummaryThe cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection

PLK1 down-regulates parainfluenza virus 5 gene expression.

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly

AKT1-Dependent Activation of NF-κB by the L Protein of Parainfluenza Virus 5▿

Innate immunity plays a critical role in the control of viral infections. The induction of innate immune responses requires activation of transcription factors. In particular, NF-κB plays an essential role in activating the expression of cytokines involved in innate immunity such as beta interferon (IFN-β) and interleukin-6 (IL-6). However, the mechanisms by which viruses activate NF-κB are poorly defined. Infection by parainfluenza virus 5 (PIV5), a prototypical member of the Paramyxoviridae family of Mononegavirales, has been shown to activate the expression of IFN-β and IL-6. To examine how PIV5 induces this expression, we have examined the activation of NF-κB by PIV5 proteins. We have found that expression of PIV5 L protein alone is sufficient to activate NF-κB. The L protein of PIV5, the catalytic component of the viral RNA-dependent RNA polymerase, contains six domains that are conserved among all negative-stranded nonsegmented RNA viruses. We have mapped the region that activates NF-κB to the second domain, which is thought to be involved in RNA synthesis. The activation of NF-κB by L requires AKT1, a serine/threonine kinase, since AKT1 small interfering RNA, an AKT inhibitor as well as a dominant-negative mutant of AKT1, blocks this activation. Furthermore, we have found that L interacts with AKT1 and enhances its phosphorylation. We speculate that L may encode AKT1 kinase activity

Activation of IFN-β expression by a viral mRNA through RNase L and MDA5

IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-β. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5′-cap and 3′-poly (A), can activate IFN expression through an RNase L-MDA5 pathway