SUMMARYUncoating of influenza virus (strain WSN) in MDCK cells was studied by following the fate of the virus labelled with radioactive precursors. The accumulation of subviral components of input virus was observed in nuclear-associated cytoplasm (NAC) obtained by treatment of the nuclei with citric acid. Two types of subviral components were found there, ribonucleoproteins (RNPs) and larger subviral particles (SVP) containing RNPs in association with M protein. SVP, with different relative amounts of M protein, were revealed in NAC, suggesting that M protein was gradually released from RNPs. The released RNPs entered the nuclei while M protein accumulated within perinuclear membranes. Thus, SVP could be regarded as probable intermediates in virus uncoating. Rimantadine prevented the release of M protein from RNPs and their penetration into the nuclei provoking the accumulation of subviral components in NAC.

SUMMARYA rimantadine-resistant variant of the Texas strain of influenza virus (Tr) was obtained by serial passages in eggs and in MDCK cells in the presence of the drug, and its uncoating in MDCK cells was compared to that of the sensitive variant (Ts). First and second steps of uncoating were defined respectively by the appearance of subviral particles (SVP) in nuclear-associated cytoplasm (NAC) and ribonucleoproteins (RNPs) in nucleoplasm. In cells infected with Ts, SVP and RNPs were revealed in NAC, while in the presence of rimantadine RNPs were neither found in NAC nor in the nucleoplasm. In cells infected with Tr, SVP but not RNPs were observed in NAC. The amount of RNPs in the nucleoplasm was almost unchanged in rimantadine-treated cells, demonstrating that rimantadine did not interfere with uncoating of the resistant variant. These findings confirm the suggestion that rimantadine blocks the second step of uncoating of sensitive influenza viruses, and are consistent with the idea that this event does account for the prevention of influenza virus infection by the drug.

SUMMARYChicken fibroblasts and MDCK cells were infected with influenza virus labelled with either aH-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 x h p.i. A part of the intranuclear parental structures was associated with chromatin while the other part could be extracted from nucleoplasm by o'16 M-NaCI and represented free ribonucleoprotein (RNP) particles. These RNPs sedimented in glycerol velocity gradients at 4o to 7oS, very similar to cytoplasmic RNPs, but differed distinctly from them in buoyant density. The bulk of cytoplasmic RNPs after fixation with formaldehyde banded in CsCI at I'34 g/ml while nucleoplasmic RNPs banded at 1.39 or I'4I 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.

Two steps of influenza virus A/WSN uncoating in MDCK cells are described. The products of the first step are cores which accumulate in the nuclear-associated cytoplasm. The products of the second step are ribonucleoproteins which penetrate the nuclei. Rimantadine blocks the second step without interfering with the first one.