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

Abstract

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.

Cellular CARD11 Inhibits the Fusogenic Activity of Newcastle Disease Virus via CBM Signalosome-Mediated Furin Reduction in Chicken Fibroblasts

“…The expression of CARD11 was not significantly induced by velogenic and lentogenic strains in the infected CEFs and DF-1 cells. However, CARD11 was able to inhibit viral replication and suppress viral polymerase activity in fibroblasts, which was consistent with the results of our previous study ( Wang et al, 2019 ). Importantly, we found that CARD11 inhibited the fusogenic activity of NDV by activating the CBM signalosome to suppress furin expression, resulting in reduced cleavage efficiency of the F protein.…”

“…The CPE was visualized under an Olympus IX73 inverted research microscope (Japan). The viral titers of F48E9-infected cells were determined by a 50% tissue culture infective dose (TCID 50 /mL) assay, while those of LaSota-infected DF-1 cells were determined by an immunofluorescence assay (IFA) in DF-1 cells ( Wang et al, 2019 ). The titer of TCID 50 /mL was calculated according to the method established by Reed and Muench ( Guo et al, 2018 ).…”

“…The minigenome (MG) system of the LaSota strain was constructed as previously described ( Wang et al, 2019 ). A 5-plasmid system of MG was used: pCAGGS-T7, pMG-Fluc, pcDNA3-HA-NP, pcDNA3-HA-P, and pCAGGS-L at a ratio (μg) of 5:5:2:2:1 that were cotransfected with pTSB-CMV-CARD11-SBP-3Flag-tRFP-F2A-Puro (2 μg) or pTSB-CMV-MCS-SBP-3Flag-tRFP-F2A-Puro (2 μg) into DF-1 cells.…”

“…Host cell proteins play essential roles in NDV pathogenesis. In our previous study, an NDV-induced brain-specific upregulated protein, caspase recruitment domain 11 (CARD11), was identified to inhibit viral replication through the CARD11 CC1 domain, and the viral L protein was found to competitively interact with the X domain of P , which hampers the P – L interaction and suppresses viral polymerase activity and viral replication ( Wang et al, 2019 ). CARD11 comprehensively exists in human and animal tissues ( Fagerberg et al, 2014 ; Yue et al, 2014 ).…”

Identification of a potential neutralizing linear epitope of hemagglutinin-neuraminidase in Newcastle disease virus

“…The animals were subcutaneously injected with a mixture of the protein and incomplete Freund’s adjuvant (Sigma) at 14 and 28 days. After the guinea pigs were anesthetized with ketamine and xylazine, their antisera were collected and stored at − 80 °C [ 24 ].…”