The basic issue to be examined is the mechanism of tissue specific gene expression and DNA replication using mouse polyomavirus. We will focus on the issue of cell specific DNA replication and its relationship to viral and cellular cell specific gene control. There are five specific aims as follows: 1. To continue the genetic analysis of tissue specific polyoma replication in vivo. We will construct further mutants to examine how point deletions within two domains of polyoma (Py) regulatory DNA (A and B enhancer) apparently broaden tissue specificity replication. In addition we will use genetic methods to examine the ability of various sub-enhancer multimers to also replicate in broader organ types. The apparent ability of the B enhancer to restrict viral replication in some but not other cell types will also be investigated. We will begin a genetic analysis of enhancer requirements for persistent infections. The in vivo analysis will be expanded to include in situ hybridization of all infected organs in order to establish the exact cell specificity within organs. 2. We will further examine the effect of Adenovirus E1A on Polyomavirus DNA replication. We will determine if E1A is inducing a stable or possibly cell cycle control of polyoma DNA replication in permissive cells. In addition, we will examine the viral and cellular proteins involved in viral DNA replication to determine if they have undergone some detectable modification. We will also attempt to establish if their is an enhancer sequence that is the target of E1a suppression or if this suppression is not via cis acting regulatory DNA. Various other E1A proteins (from murine adenovirus, mutants in conserved regions) will also be examined to determine if they affect the repression of Py DNA replication we see. 3. The relationship of terminal differentiation to amplified Py DNA replication be further studied. Various primary and established cell lines which can be induced to terminally differentiate will be examined for their ability to replicate wt and enhancer mutant polyomavirus and the generality of the relationship of terminal differentiation to DNA formation and episomal persistence in undifferentiated cells. If a general relationship is confirmed (ie all terminal lines are more permissive for Py DNA replication), a genetic analysis will be done to determine if common regulatory sequences are involved and if distinct but common cellular replication or chromatin proteins are also involved. 4. We will do a more systematic genetic analysis of the sequence requirements for cell specific DNA replication relative to transcription. We will examine the spacing and heterodimer issue of transcriptional elements and better define the elements required for if replication relative to transcription in specific cell types. We will also examine if cell type affects the spacing relationship of enhancer to the origin of replication. 5. Finally, we will attempt to establish an in vitro system for the analysis of cell specific polyoma DNA replication. Initially we will conduct footprinting experiments to determine relative quantities and binding affinities of cellular factors which appear to participate in cis regulated initiation of viral DNA replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036605-09
Application #
2178454
Study Section
Virology Study Section (VR)
Project Start
1986-04-01
Project End
1995-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
9
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Villarreal, L P; Defilippis, V R; Gottlieb, K A (2000) Acute and persistent viral life strategies and their relationship to emerging diseases. Virology 272:6-Jan
Gottlieb, K; Villarreal, L P (2000) The distribution and kinetics of polyomavirus in lungs of intranasally infected newborn mice. Virology 266:52-65
Piatti, P G; Gottlieb, K A; Taylor, J A et al. (1998) Approaches to study interactions between small DNA viruses and differentiated tissue. Methods 16:62-82
You, Z; Villarreal, L P (1996) Mapping of polyomavirus DNA replicative intermediates by two-dimensional gel analysis using chemiluminescent detection. J Virol Methods 56:173-8
Atencio, I A; Belli, B; Hobbs, M et al. (1995) A model for mixed virus disease: co-infection with Moloney murine leukemia virus potentiates runting induced by polyomavirus (A2 strain) in Balb/c and NIH Swiss mice. Virology 212:356-66
Shadan, F F; Villarreal, L P (1995) The evolution of small DNA viruses of eukaryotes: past and present considerations. Virus Genes 11:239-57
Atencio, I A; Meraz, A; Villarreal, L P (1994) The secretory cells of mouse salivary glands are nonpermissive for polyomavirus replication. Virology 200:842-8
Atencio, I A; Villarreal, L P (1994) Polyomavirus replicates in differentiating but not in proliferating tubules of adult mouse polycystic kidneys. Virology 201:26-35
Shadan, F F; Cowsert, L M; Villarreal, L P (1994) n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses. J Virol 68:4785-96
Atencio, I A; Shadan, F F; Zhou, X J et al. (1993) Adult mouse kidneys become permissive to acute polyomavirus infection and reactivate persistent infections in response to cellular damage and regeneration. J Virol 67:1424-32

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