Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein

Qiu, Xusheng; Zhan, Yuan; Chen, Meng; Wang, Junqing; Dong, Luna; Sun, Yingjie; Tan, Lei; Chen, Song; Yu, Shuang; Ding, Chan

doi:10.1007/s00705-016-2884-x

Cited by 15 publications

(12 citation statements)

References 49 publications

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“…10D and E) but not the CARD/LATCH domain ( Fig. 10F), indicating that only the CC1 domain of CARD11 interacted with the viral P. Correspondingly, P was divided into four regions (NP 0 -binding region, disordered region, PMD, and X domain) according to a previous report (31). To identify which domain of P interacts with CARD11, we constructed P truncations (Fig.…”

Section: Hek293a Cells and Chpncsmentioning

confidence: 88%

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Wang

Chang

Yao

et al. 2019

J Virol

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Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-κB signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species. IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

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Section: Hek293a Cells and Chpncsmentioning

confidence: 88%

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Wang

Chang

Yao

et al. 2019

J Virol

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“…The role of phosphorylation of viral proteins in the viral live cycle has been increasingly recognized, as demonstrated for multiple viruses, including influenza A virus (39), Newcastle disease virus (40), hepatitis C virus (41), dengue virus (42), and Rift valley virus (43), among others. While phosphatases are involved in multiple normal physiological reactions of cells, targeting of their noncatalytic sites, which prevents their interaction with only certain substrates, including viral proteins, may be a viable approach to develop broadly specific antivirals.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription

Tigabu

Ramanathan

Ivanov

et al. 2018

J Virol

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The filoviruses Marburg (MARV) and Ebola (EBOV) cause hemorrhagic fever in humans and non-human primates with case high fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation at amino acid residues 46-53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, co-immunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule 1E7-03 targeting a non-catalytic site of the enzyme, which previously showed to increase EBOV VP30 phosphorylation, was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at the cluster of phosphorylated amino acids Ser-46-Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35 and dramatically reduced replication of infectious MARV particles. Thus MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. The largest outbreak of MARV occurred in Angola in 2004-2005 and caused 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of MARV genome demonstrated differences compared to EBOV. The identified phosphorylation sites appeared to inhibit transcription, and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility of development of antivirals based on compounds targeting PP1.

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“…Plasmids-transfected BSR-T7/5 cells or viruses-infected DF-1 cells were collected and then treated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA was extracted and reverse-transcribed (2 μg per sample) as described previously [27]. Quantification of minigenomic RNA synthesis [27] and viral RNA synthesis [16] by qRT-PCR was performed as previously described, respectively.…”

Section: Methodsmentioning

confidence: 99%

Nuclear localization of Newcastle disease virus matrix protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription

Duan

Deng

et al. 2019

Vet Res

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Nuclear localization of paramyxovirus proteins is crucial for virus life cycle, including the regulation of viral replication and the evasion of host immunity. We previously showed that a recombinant Newcastle disease virus (NDV) with nuclear localization signal mutation in the matrix (M) protein results in a pathotype change and attenuates viral pathogenicity in chickens. However, little is known about the nuclear localization functions of NDV M protein. In this study, the potential functions of the M protein in the nucleus were investigated. We first demonstrate that nuclear localization of the M protein could not only promote the cytopathogenicity of NDV but also increase viral RNA synthesis and transcription efficiency in DF-1 cells. Using microarray analysis, we found that nuclear localization of the M protein might inhibit host cell transcription, represented by numerous up-regulating genes associated with transcriptional repressor activity and down-regulating genes associated with transcriptional activator activity. The role of representative up-regulated gene prospero homeobox 1 (PROX1) and down-regulated gene aryl hydrocarbon receptor (AHR) in the replication of NDV was then evaluated. The results show that siRNA-mediated knockdown of PROX1 or AHR significantly reduced or increased the viral RNA synthesis and viral replication, respectively, demonstrating the important roles of the expression changes of these genes in NDV replication. Together, our findings demonstrate for the first time that nuclear localization of NDV M protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription, improving our understanding of the molecular mechanism of NDV replication and pathogenesis.Electronic supplementary materialThe online version of this article (10.1186/s13567-019-0640-4) contains supplementary material, which is available to authorized users.

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Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein

Cited by 15 publications

References 49 publications

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription

Nuclear localization of Newcastle disease virus matrix protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription

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