Molecular Biology of Retroviruses, Immunology, cancer biology, gene therapy
Professor Fan's laboratory is investigating mouse retroviruses, by molecular biological techniques, as model systems for regulation of gene expression, chromatin structure, and carcinogenesis. During infection, retroviruses synthesize a DNA copy of the viral RNA genome using the viral enzyme reverse transcriptase. The viral DNA is then integrated into the host chromosomal DNA. This integrated viral DNA is a complete transcription unit that is transcribed by cellular RNA polymerase. As such, retroviruses offer a convenient system for studying eukaryotic gene expression. Manipulations in viral DNA can be performed by molecular cloning techniques, and the altered viruses can be introduced into cells by DNA transfection.
Current experiments are focused on the viral long terminal repeats (LTR's) which contain the signals for initiation and termination of viral RNA synthesis. A series of LTR deletion mutants have been constructed, and other cellular and viral regulatory sequences have also been inserted. These alterations change both viral infectivity and pathogenicity and they are being analyzed in a systematic fashion. Since enhancers have been implicated in the tissue specificity of gene expression, one region of particular interest is the transcriptional enhancer sequences in the viral LTR.
Another line of experimentation involves study of the mechanism by which Moloney murine leukemia virus induces T-lymphoma in mice. An M-MuLV enhancer variant (Mo+PyF101 M-MuLV) has been useful, since this mutant does not efficiently induce disease. Studies of wild-type and Mo-PyF101 M-MuLV-innoculated animals led to the identification of a virus-induced preleukemic state, characterized by generalized hematopoietic hyperplasia in the spleen. Recent experiments indicate that this hyperplasia is an indirect consequence of viral infection, probably secondary to a hematopoietic defect in the bone marrow. Additional experiments implicate 'MCF' envelope gene recombinants (arising in the mouse by recombination between innoculated M-MuLV and endogenous MuLV- related genetic material) in development of preleukemic hyperplasia.
A third line of work concerns development of advanced assay systems for chemical carcinogens. Currently, a significant percentage of chemical carcinogens are not detected by standard short-term mutagenesis (Ames) assays in Salmonella. One possible solution is to over-express normal cell proto-oncogenes in cells, since this might render them more sensitive to transformation by genotoxic or non-genotoxic chemicals. Retroviral vectors expressing various proto-oncogenes (e.g., murine c-myc and rat neu) were generated and introduced into Rat 6 and mouse C2H10T1/2 cells. Overexpression of c-myc in these cells increased their transformability by two model carcinogens, benzo(a)pyrene and MNNG. Individual clones with heightened transformation responses will be tested for transformation by an expanded collection of genotoxic and non-genotoxic carcinogens (and noncarcinogens). Such cells might form the basis for new carcinogen detection systems.
Early infection and spread of a conditionally replicating adenovirus under conditions of plaque formation.
Hofacre A, Wodarz D, Komarova NL, Fan H.
Virology. 2012 Feb 5;423(1):89-96. Epub 2011 Dec 20.
Cell transformation by RNA viruses: an overview.
Viruses. 2011 Jun;3(6):858-60. Epub 2011 Jun 15. No abstract available.
Insertional oncogenesis by non-acute retroviruses: implications for gene therapy.
Fan H, Johnson C.
Viruses. 2011 Apr;3(4):398-422. Epub 2011 Apr 15.
Jaagsiekte sheep retrovirus biology and oncogenesis.
Hofacre A, Fan H.
Viruses. 2010 Dec;2(12):2618-48. Epub 2010 Dec 3
Johnson, C., Sanders, K., and Fan, H. Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture. J of Virol. 2010. 84(10): 5379-5390.
Nitta, T., Kuznetsov, Y., McPherson, A., and Fan, H. Murine Leukemia Virus Glycosylated Gag (gPr80 gag) Facilitates Interferon-Senstive Virus Release Through Lipid Rafts. PNAS. 2010. 107(3) 1190-1195.
Nitta, T., A. Hofacre, S. Hull, and H. Fan. Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA. J Virol. 2009. 83:12499-511.
Hofacre, A., T. Nitta, and H. Fan. Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein. J Virol. 2009. 83:12483-98.
Okeoma, C.M., Low, A., Bailis, W., Fan, H.Y., Peterlin, B.M., and Ross, S.R. Induction of APOBEC3 in vivo Causes Increased Restriction of Retrovirus Infection. J. Virol. 2009. 83:3486-3495.