Paramyxovirus persistence
Abstract
In this study, SV5 infection of Balb/c mouse fibroblast cells has been used as a
model system to investigate the possible mechanisms underlying the establishment and
maintenance of Paramyxovirus persistence.
It was found that following entry to these cells, the virus initiated a wave of
transcription and replication, similar to that of a permissive infection, in which normal
levels of each of the virus proteins were synthesized. However, by 48-72 hours post
infection (p.i.) there was an almost complete cessation of virus mRNA and protein
synthesis. Despite the decrease in virus activity, full length viral genome RNA and P and
NP, the proteins involved in transcription and replication, could be detected at consistently
high levels up to 5 days p.i., although the levels of HN, M, F and V declined.
Immunofluorescence analysis supported these data showing that at later times p.i.
although there were some cells positive for all the viral proteins, a high proportion of cells
were strongly positive for NP, L and P, but negative for M, F and HN. In these cells, NP, L
and P were often located in discrete cytoplasmic foci. These results suggested that the
persistently infected cell population consisted of some cells in which the virus was active
and other in which it was quiescent within cytoplasmic inclusions.
A series of cell lines was established from a monolayer of Balb/c cells that had been
infected at a high multiplicity. Immunofluorescence studies showed only a minority of
cells in these clones to be infected with virus, indicating that during division, not all
daughter cells became infected. Of the infected cells, some were positive for all the viral
proteins, while others were positive for only NP and P. Co-cultivation of the cloned cells
with Vero cells, which are permissive for SV5 replication, rapidly yielded non-defective
virus, suggesting that the virus was active in some cells. These results suggested that the
persisting virus was in a state of flux, able to reside as inclusions of inactive nucleocapids
from which it could reactivate to initiate a new round of infection.
Experiments aiming to determine if the persistently infected cells were resistant to
immune attack demonstrated that cells at 5 days p.i., in which the majority of cells were
quiescently infected, were less susceptible to immune lysis than cells at 1 day p.i. in which
there was ongoing protein synthesis.
Further experiments were carried out both to try to determine what had induced the
persistent state in mouse cells and also to examine factors which might induce a similar
state in different cell lines.
Type
Thesis, PhD Doctor of Philosophy
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