Cytoplasmic and nuclear input virus RNPs in influenza virus-infected cells

Chicken fibroblasts and MDCK cells were infected with influenza virus labelled with either 3H-uridine or 14C-amino acids, and the location in infected cells and properties of input virus-labelled structures were studied. Input virus RNA and protein were found in the cytoplasm of nuclei 1 h p.i. A part of the intranuclear parental structures was associated with chromatin while the other part could be extracted from nucleoplasm by 0.16 M-NaCl and represented free ribonucleoprotein (RNP) particles. These RNPs sedimented in glycerol velocity gradients at 40 to 70S, very similar to cytoplasmic RNPs, but differed distinctly from them in buoyant density. The bulk of cytoplasmic RNPs after fixation with formaldehyde banded in CsCl at 1.34 g/ml while nucleoplasmic RNPs banded at 1.39 or 1.41 g/ml. RNPs isolated from virions and infected cells contained the NP polypeptide which was revealed by SDS-PAGE analysis as a double band. The ratio of the two bands varied in cytoplasmic and nucleoplasmic RNPs, the lower band being dominant in cytoplasmic but not in nucleoplasmic RNPs. In addition, cytoplasmic RNPs were phosphorylated. The possible significance of intracellular RNP modifications for virus replication is discussed.     

Identification of human Sm and (U1) RNP antigens by immunoblotting

When HeLa nuclear extracts or ribonucleoproteins (RNPs) from rat liver nuclei were used as antigens, a monospecific anti-(U1)RNP serum recognized in each preparation only 1 polypeptide of 68 or 70 kilodalton (kd) respectively. With a serum of combined anti-Sm/(U1)RNP specificity, HeLa nuclear extracts showed 3 additional antigenic polypeptides of 29, 28, and 16 kd, whereas only 2 additional polypeptides of 27 and 16 kd were observed in rat liver RNPs. However, no antigenic reaction at 68/70 kd was detected with a monospecific anti-Sm serum, indicating that the 68/70 kd antigen is specific for anti-(U1)RNP antibodies. When commercially available ENA extract was used as antigen source only weak immunostaining in the range 70-40 kd and at 16 kd was seen. Elution experiments with anti-Sm antibodies bound to their specific polypeptides demonstrated that neither protein degradation nor cross-reaction was responsible for recognition of the 29/28 and 16 kd antigens by this serum, and that in fact 2 different autoantibody systems are involved.