- Volume 68, Issue 6, 1987

We describe here a persistently infected cell system with unique properties. Cells infected with human parainfluenza virus type 3 (PF3) at high multiplicities of infection showed little or no cytopathic effects (cell fusion). Unlike other paramyxovirus persistent infections that require a long development time, the majority of the cells survived the initial infection and formed persistently infected cell cultures that were immediately available for study. In addition, unlike other paramyxovirus persistent infections, the PF3 system described here produced high levels of infectious virus and did not undergo periodic crises. Although cells persistently infected with PF3 contained large amounts of the cleaved, active form of the viral fusion protein, F, the persistently infected cells did not fuse with each other. However, they did fuse with uninfected cells within minutes of cell-to-cell contact. Other persistent paramyxovirus infections do not have this property. Fusion occurred with all cells tested, including red blood cells, and was not dependent on protein synthesis. The unique fusion properties of these PF3 persistently infected cells make this an interesting system for the study of mechanisms of viral fusion and mechanisms of inhibition of viral fusion.

The ability of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) to replicate in peripheral blood monocytes cultured in vitro for 1, 2, 4 or 7 days prior to infection was investigated. Inoculation of 1-day old monocytes produced at least tenfold less new virus than infection of the older, more macrophage-like cells for both viruses. PIV3 induced extensive syncytium formation, whereas RSV caused a cytopathic effect manifest by increased rounding of the cells with minimal syncytium formation. Supernatants of infected monocytes were assayed for human interferon-a (HuIFN-α) in an attempt to explain the restricted viral replication in the youngest monocytes. In PIV3-infected cells, HuIFN-α production was inversely correlated with new virus formation. Monocytes infected after 1 day in culture produced 800 IU/ml of HuIFN-α; the older cells produced 100 to 200 IU/ml. In contrast, monocytes infected on day 1 with RSV produced minimal amounts (1·5 IU/ml) of HuIFN-α. Increasing amounts of HuIFN-α were detected in cells infected with RSV after 2, 4 or 7 days in culture, reaching a maximum of 400 IU/ml on day 7. Further investigation of the apparent restriction of replication in young monocyte cultures may be helpful in understanding the pathogenesis of these respiratory infections.

Cultured spinal ganglia and cord from mice and hamsters were infected with mumps virus or Sendai virus. Expression of five structural proteins, the haemagglutinin-neuraminidase, fusion, nucleocapsid (NP), phospho (P) and matrix proteins was examined with monoclonal antibodies to each protein. In Sendai virus-infected mouse neurons all five viral proteins were detected. In hamster neurons infected with mumps virus all viral proteins were also expressed, but in mouse neurons only the NP and P proteins were seen. This suggests a species-specific cellular restriction of viral protein synthesis in mumps virus-infected mouse neurons. There was no, or only a slight, reduction in the number of neurons between days 4 and 20 after infection of mouse cultures with mumps virus, but the proportion of infected neurons diminished from 68% to 15% during this time.

The methylation pattern of herpes simplex virus type 1 (HSV-1) DNA, present in the central nervous system of latently infected mice, was examined by digestion of the DNA with methylation-sensitive restriction endonucleases and Southern blot hybridization. Using the enzymes SmaI, XmaI, SalI and SacII, the data indicate no extensive methylation of latent HSV-1 DNA in vivo. Thus, extensive methylation of the viral genome is not a necessary condition for, or a consequence of maintaining, the latent state in vivo.

Transcription in vitro by the RNA polymerase of infectious haematopoietic necrosis virus (IHNV), a salmonid rhabdovirus, was investigated using different reaction conditions to maximize RNA synthesis. The use of HEPES buffer rather than Tris buffer, and the addition of S-adenosyl-l-methionine to the reactions resulted in a sixfold increase in RNA synthetic activity to 6400 pmol UMP incorporated/mg viral protein/hour. The RNA transcripts produced in this system contained polyadenylated species which co-migrated with IHNV mRNA species 2, 3, 4 and 5 from IHNV- infected cells. The transcripts were shown to be functional mRNA species by their ability to direct the synthesis of viral proteins in vitro.

RNA from clover yellow mosaic virus, a potexvirus, was translated in rabbit reticulocyte lysates, and in vitro translation products were compared with virus-specific proteins produced in vivo in pea mesophyll protoplasts inoculated with viral RNA. One major polypeptide with an M r of 182000 (182K) was produced in vitro, and two were produced in vivo, with M r 182K and 22K. The 182K product was thought to be the same in vitro as in vivo; the 22K product was shown by Western blotting to be the virus coat protein.

The 32000 mol. wt. non-structural protein (P3) of alfalfa mosaic virus (A1MV) has previously been shown to accumulate in the cell wall fraction of tobacco leaves infected with AlMV. We now report the ultrastructural location of this protein. P3 was visualized immunocytochemically in the middle lamella of the walls of those parenchymal or epidermal cells which had just been reached by the infection front and in which viral multiplication had just begun. P3 was not found when AlMV had accumulated to high levels in infected cells. These findings support the concept that P3 is involved in the spread of viral infection from cell to cell, i.e. is the transport factor of AlMV.