Haixu Xu

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process.

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.

Surveillance of avirulent Newcastle disease viruses at live bird markets in Eastern China during 2008–2012 reveals a new sub-genotype of class I virus

BackgroundThe strains of Newcastle disease virus (NDV) can be divided into two distinct clades: class I and class II. At present, limited molecular epidemiological data are available for the class I virus at live bird markets (LBMs). Knowing the genomic and antigenic characteristics of class I NDVs might provide important insights into the evolution dynamics of these viruses. In this study class I NDVs isolated from LBMs in Eastern China between 2008 and 2012 were characterized.ResultsWe characterized 34 class I NDVs genetically and 15 of the 34 NDVs pathologically which originated from geese, chickens and ducks at live bird markets. Based on the older classification system, twelve of fourteen strains isolated from 2008 to 2010 belonged to sub-genotype 3b. However, the rest 22 strains formed a separate novel cluster in genotype 3, which was designated as sub-genotype 3c. When based on the new classification system, sub-genotype 3b was classified into sub-genotype 1a and the sub-genotype 3c was classified into sub-genotype 1b. Over 62% (21/34) of the viruses were chicken-origin and only 13 isolates were waterfowl-origin. The Cross-neutralization reactions between CK/JS/05/11, CK/JS/06/12 and the vaccine strain LaSota showed significant antigenic differences between them.ConclusionsCurrently, sub-genotype 3c (or 1b) NDVs are the most frequently isolated classI strains at LBMs in Eastern China., and the class I NDVs has transferred from waterfowls to chickens and circulated in chicken flocks extensively.

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.

A single amino acid mutation, R42A, in the Newcastle disease virus matrix protein abrogates its nuclear localization and attenuates viral replication and pathogenicity

The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 aa of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted Ms nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.

The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1 clade 2.3.4 avian influenza virus in guinea pigs

We generated and characterized site-directed HA mutants on the genetic backbone of H5N1 clade 2.3.4 virus preferentially binding to α-2,3 receptors in order to identify the key determinants in hemagglutinin rendering the dual affinity to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors of the current clade 2.3.4.4 H5NX subtype avian influenza reassortants. The results show that the T160A substitution resulted in the loss of a glycosylation site at 158N and led not only to enhanced binding specificity for human-type receptors but also transmissibility among guinea pigs, which could be considered as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.

The M, F and HN genes of genotype VIId Newcastle disease virus are associated with the severe pathological changes in the spleen of chickens.

BackgroundThe strains of the genotype VIId Newcastle disease virus (NDV) induce more severe tissue damage in lymphoid organs than other virulent strains. The underlying molecular mechanisms are poorly understood.MethodsGenotype IV NDV Herts/33 and genotype VIId NDV JS5/05 have a distinctive pathological profile in the spleen. These two strains of viruses were selected as parental viruses to generate a panel of chimeric viruses by replacing the M, F and HN genes of Herts/33 individually or in combination with the corresponding genes of JS5/05 using reverse genetic. Virulence and in vitro characteristics of the recombinant viruses were assessed. In addition, pathological changes, virus load, and transcriptional cytokine response in the spleen of chickens infected with these recombinant viruses were also analyzed.ResultsPathogenicity test showed that all chimeric viruses are virulent. In vitro characterization revealed that gene replacement did not change growth kinetics and HN expression on cell surface of the recombinant viruses. However, replacement of the M, F and HN genes resulted in apparent changes in the fusion activity. Moreover, pathological studies revealed that only inclusion of the homologous M, F and HN genes of JS5/05 in Herts/33 backbone resulted in severe pathological changes characterized by extensive necrosis in the spleen, similar to that induced by JS5/05. In addition, this gene replacement significantly increased virus replication and the levels of transcriptional cytokine response, compared to Herts/33. Conversely, inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone resulted in Herts/33-specific pathological changes and significantly decreased virus load and the expression levels of cytokine genes, compared to JS5/05.ConclusionsThe M, F and HN genes are related to the severe pathological changes in the spleen of chickens infected with genotype VIId NDV.

Characterization of the nuclear import pathway for BLM protein

Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin β1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin β1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin β1 through its NLS domain with the 14-16 HEAT repeats of importin β1. Furthermore, importin β1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin β1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.

Simultaneous mutation of G275A and P276A in the matrix protein of Newcastle disease virus decreases virus replication and budding

The matrix (M) protein of Newcastle disease virus (NDV) is a highly conserved hydrophobic viral protein. In some paramyxoviruses (measles virus and Sendai virus), the paired glycine (G) near the C terminus of the M protein may form a turn that mediates the specific interaction with the cell membrane. Similar amino acids (glycine-proline [GP], at position 275–276) exist in the M protein of NDV. However, the role of these residues in the replication and pathogenicity of NDV is unknown. In this study, recombinant NDV with the sequence GP/AA or LGP/GGL in the M protein was generated to investigate the role of this conserved sequence. Budding experiments on the mutant viruses revealed that the GP/AA mutation reduced virus budding and virus replication in DF-1 cells; biological characterization revealed attenuated virulence and pathogenicity in chickens, indicating that the GP sequence plays a critical role in the life cycle of the virus.

Two amino acid substitutions in the haemagglutinin of the 2009 pandemic H1N1 virus decrease direct-contact transmission in guinea pigs.

The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the haemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the α2,6-sialoglycan to the α2,3-sialoglycan receptor, and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.