Neuronal cell model and methods of use therefor

Details Neuronal cell model and methods of use therefor Neuronal cell model and methods of use therefor

1999-03-19

2001-07-31

Wortman, Donna C. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S353000

Reexamination Certificate

active

06268124

ABSTRACT:

FIELD OF THE INVENTION
In this disclosure, it is demonstrated that neurally-differentiated cells can be infected with viruses in a manner that supports a long-term non-productive infection. This model when compared to others is unique in that 1) quiescent infection is established in a neuronal cell line that has features reminiscent of ganglionic neurons, 2) an inhibitor of DNA synthesis is needed only to establish the quiescent state, not maintain it, 3) an inhibitor of viral gene expression is not required to establish the quiescent state, 4) the non-productive state is reversible with spontaneous and inducible reactivation, and 5) quiescence is long term.
In particular, neuronally differentiated PC12 cells, established from pheochromocytoma of rat adrenal medulla, are used to host a persistent viral infection. First, cells are differentiated by growing cells in defined medium containing nerve growth factor. Under these conditions cells extend neurites, develop electrical excitability and express genes encoding neuronal cell-specific proteins (Green and Tischler 1976; Green and Tischler 1982). Cells are maintained in serum-free medium to render them nondividing. Next, cells are inoculated with neurotropic virus (eg., human herpes virus) under conditions that restrict viral propagation. A defined regimen of media changes are used to establish a quiescent and nonproductive state following the withdrawal of the antiviral treatment. Evidence of establishment of a quiescent and persistent infection comes from assays demonstrating that cells survive the infection, a nonproductive viral state is established in the majority of cultures, and cells support spontaneous and inducible virus production.
BACKGROUND OF THE INVENTION
The lack of a universally accepted neural cell-line that supports viral latency, in particular, HSV-1 latency, has restricted the understanding of the molecular events involved in reactivation from latency. As a result, animals and tissue culture have served to provide an understanding of the mechanisms of this event. Animal models, however, are limited by difficulties. These include: (i) latency and reactivation events that are influenced by viral strains with different primary growth phenotypes, (ii) the limited number of neurons latently infected in animal models (Bloom et al, 1996; Hill et al, 1996; Maggioncalda et al, 1996; Mehta et al, 1995; Ramakrishnan 1994; Sawtell, 1997; Sawtell et al, 1998; Thompson and Sawtell, 1997), and (iii) inaccurate quantitation of reactivation events when measuring virus production at the recurrent site as a result of influences of transport, replication in epithelium, and the immune response.
A major advantage of tissue and cell culture models includes the ability to observe virus at the single cell level without the overlay of immunological events that modulate the eventual appearance of virus in the host. Tissue culture models derived from neuronal and sympathetic ganglia have properties of the in vivo system including: (i) restricted transcription of the HSV genome (Doerig et al, 1991; Halford et al, 1996; Smith et al, 1992; Smith et al, 1994), (ii) lack of virus production following removal of the inhibitory agent, (Wilcox and Johnson, 1988) (iii) the presence of latency-associated transcripts (LATs) (Doerig et al, 1991; Smith et al, 1994), (iv) impaired reactivation of thymidine kinase negative virus (Wilcox et al, 1992), and (v) inducible reactivation (Halford et al, 1996; Moriya et al, 1994; Smith et al, 1992; Wilcox and Johnson, 1988; Wilcox and Johnson 1987; Wilcox et al, 1990). Nevertheless, preparation of dissected ganglia is inconvenient, material is limited, animal use is required, and axotomy introduces traumatic factors that influence reactivation of virus.
For the above reasons, cell culture models are important for studying the molecular details of the establishment, maintenance and reactivation stages of latency. Cell culture also allows for an unlimited supply of a defined host cell and the ability to manipulate genetic material. Over the past 25 years, cell culture systems using fibroblast cultures (Harris and Preston, 1991; Jamieson et al, 1995; O'Neill, 1977; O'Neill et al, 1972; Russell et al, 1987; Scheck et al, 1989; Wigdahl et al, 1982a; Wigdahl et al, 1982b; Wigdahl et al, 1983) and lymphocytes (Hammer et al, 1981; Youssoufian et al, 1982) have enabled the study of HSV-1 during a latent-like state.
These models, however required low input multiplicities and/or the use of replication inhibitors such as anti-viral agents, inhibitory temperatures, or the use of a mutant virus, to prevent virus production. A cell line that has neural morphology and physiology, can survive infection and permit viral production, allow establishment of a long term nonproductive viral infection, and support virus in a state suitable for reactivation studies would be more desirable.
The rat pheochromocytoma (PC12) cell line (used in the present model) is of neural crest origin and can be morphologically differentiated with the addition of nerve growth factor (Greene and Tischler, 1976). These cells have been shown to be permissive to HSV-1 infection (Bloom and Stevens, 1994; Rodahl and Haar, 1997; Rubenstein and Price, 1983a; Rubenstein and Price 1983b, Rubenstein and Price, 1984), and have been used to examine HSV-1 gene regulation and expression (Frazier et al, 1996a; Frazier et al, 1996b; Jordan et al, 1998; Leib et al, 1991; Xie et al, 1996) and the function of HSV origins of DNA replication (Hardwicke and Schaffer, 1997). The relevance of these studies, however remains incomplete since the ability of these cells to harbor HSV-1 in a “latent-like” state has not been demonstrated (Block et al, 1994). Block also differs from the present invention in that Block asserted that withdrawal of NGF from a serum-containing medium resulted in reactivation, implying that NGF and serum were necessary in any such model.
Previous studies have demonstrated NGF-differentiated PC12 (Nd-PC12) cells can be maintained as non-dividing cultures both in the presence and absence of serum (Greene, 1978; Greene and Tischler, 1976). These cultures have been studied on non-coated (Block et al, 1994) and collagen coated dishes (Greene and Tischler, 1976). Other studies indicate that a significant portion of PC12 cells cultured in the presence of serum continue to divide (Goodman et al, 1979; Ignatius et al, 1985).
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