Abstract
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.
Highlights
Paramyxoviruses describe a family of non-segmented negative-sense RNA viruses (NNSV) responsible for significant human and animal diseases, such as measles virus (MeV), mumps virus (MuV), Nipah virus (NiV), Hendra virus (HeV), Sendai virus (SeV), parainfluenza virus types 1–5, and Newcastle disease virus (NDV) [1].The RNA genomes of paramyxoviruses are 15–19 kb in length and contain six to ten genes that encode six structural viral proteins, including fusion protein (F), attachment protein (HN or H or G), nucleocapsid protein (N or NP), phosphoprotein protein (P), large polymerase protein (L), matrix protein (M) [2, 3]
We found that the cytopathic effect (CPE) and green fluorescent protein (GFP) expression in rSS1GFP-infected cells started early at 6 hpi and the extensive CPE and GFP expression appeared at 18 hpi, and the cell monolayer was absolutely destroyed at 36 hpi (Figure 1C)
The slight CPE and GFP expression in rSS1GFP-M/NLSm infected cells started at 12 hpi and the cell monolayer was still existent at 36 hpi (Figure 1C), demonstrating that rSS1GFP-M/ NLSm induced much slighter CPE and GFP expression than that of rSS1GFP
Summary
The RNA genomes of paramyxoviruses are 15–19 kb in length and contain six to ten genes that encode six structural viral proteins, including fusion protein (F), attachment protein (HN or H or G), nucleocapsid protein (N or NP), phosphoprotein protein (P), large polymerase protein (L), matrix protein (M) [2, 3]. Of all these proteins, the M protein is the most abundant protein in the virions and forms an outer protein shell around the nucleocapsid, constituting the bridge between the nucleocapsid and viral envelope [4]. The detailed functions of M protein in the nucleus has only been clarified in some NNSV such as human respiratory syncytial virus (HRSV) [8], vesicular stomatitis virus (VSV) [9, 10], and MeV [11], but the precise functions of M’s nuclear localization of NDV and other paramyxoviruses remains enigmatic
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