Newcastle Disease Virus Matrix Research Articles

Study of the influence of salt concentration on Newcastle disease virus matrix protein aggregation.

The aggregation process of Newcastle disease virus matrix protein (M protein) has been studied using light scattering. We observed that the aggregation of M protein is inversely correlated with ionic strength and that this process can be reversed by high salt concentrations. It was found that the oligomeric structure of NDV matrix protein is different from those described earlier for other matrix proteins of enveloped viruses.

The Matrix Protein of Newcastle Disease Virus Localizes to the Nucleus via a Bipartite Nuclear Localization Signal

The Newcastle disease virus matrix (M) protein expressed from a cDNA clone is observed in the nucleus of transfected cells, displaying a localization pattern identical to that observed in virus-infected cells. To identify the nuclear localization signal (NLS) in the Mprotein, M gene mutants encoding deletion and amino acid substitution proteins were constructed and expressed transiently in COS-1 cells. Protein products were examined for intracellular localization using indirect immunofluorescence. Two basic amino acid clusters in the M protein were found to be required for nuclear localization since deletion of these basic clusters or substitution with random amino acids resulted in cytoplasmic localization. Substitution of pairs of basic amino acids with non-basic residues revealed that components from both basic regions are required for nuclear localization. This interdependence between two basic clusters suggests that the NLS in the M protein belongs to the newly described class of "bipartite" NLSs. Unlike most NLSs, Mprotein sequences containing the critical basic amino acid clusters fused to two different cytoplasmic reporter proteins failed to transport these proteins to the nucleus.

Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography

The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by “chymotryptic-like” cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.

Differential detergent treatment allows immunofluorescent localization of the Newcastle disease virus matrix protein within the nucleus of infected cells

Paramyxoviruses are cytoplasmic viruses and presumably do not require any nuclear function for their replication. However, recent studies using monoclonal antibodies directed against the Newcastle disease virus matrix (M) protein have found a large portion of the M protein apparently associated with the nucleus of infected cells. Whether the M protein is associated with the cytoplasmic surface of the nucleus or whether the M protein is actually located within the nucleus has not been clearly determined. To examine this question, conditions for selectively permeabilizing the cytoplasmic membrane were sought. After treating fixed cells with a low concentration (0.02%) of the nonionic detergent Triton X-100, the cytoplasmic antigen vimentin was stained with a monoclonal antibody, but nuclear antigens were not. Apparently, 0.02% Triton permeabilizes the plasma membrane while leaving the nuclear membrane intact. Under these conditions, monoclonal antibodies directed against the NDV phosphoprotein and hemagglutinin/neuraminidase glycoprotein stained infected cells, but a monoclonal antibody to the M protein did not. The inability of the anti-M monoclonal antibody to stain the nucleus, even though the outer nuclear membrane is accessible under these conditions, indicates that the M protein is not associated with the outer membrane of the nucleus. The nuclei of infected cells treated with a higher concentration (0.05%) of Triton X-100 were stained both with antibodies to nuclear antigens and with the anti-M monoclonal antibody.